Mechanism of retained placenta and its treatment by plant medicine in ruminant animals in Oromia, Ethiopia

Retained placenta is the abnormal condition in animal reproduction in which fetal membrane failed to detach from maternal uterine wall after postpartum. The retention of placenta can create some disorders within reproductive organ of animals by allowing microorganisms to grow inside the uterus causing inflammation of uterus, fever, weight loss, decreasing milk yield, longer calving intervals and if the case is severe animals may die. The pathogenesis pathway of retained placenta development commences with an imbalance of antioxidant or oxidant capacity, decrease in estrogen production, decreased prostaglandin F2 alpha, and accumulation of arachidonic and linoleic acids in the placental tissue. Retained placenta can be treated with different methods such as manual removal, administration of intrauterine antibiotics, administration of hormonal therapy, and the use of plant medicine. The most common parts of plants used for drugs preparation are the leave, bark, root, stem and seed of the specific plant. The route of drug administration to animal is oral and vagina infusion. The herbal or plant prescriptions for treatment of placenta retention are characterized as antibiosis, antiphlogosis, immune enhancement and fertility improvement without endometrial injuries.  The plants which have been used as treatment of retained placenta in Ethiopia are Flax seed, Vernonia amygdalia, Dodonea angustifolia, Solanum acaule, Solanum acuminatum, Dovyalis spp., Galinsoga quadriradiata, Plumbago zeylanica, Momordica spp., Colocasia esculenta, Bryophyllum pinnatu and Urera hypselodendron. The main objective of this paper is to illustrate the mechanism of retained placenta and its treatment using plant medicine in current status which helps the pastoral and clinician at remote areas and also to give enough information on the plant species that are sources of different chemical ingredient for future production of modern drug treatment for retained placenta. 
 
 Key words: Antibiosis, antiphlogosis, ethinoveterinarian, retained of placenta, treatment.

detach from maternal uterine wall after postpartum.It is also called retention of fetal membrane (RFM).Retention of placenta (ROP) occurs when fetal membrane fail to detach after postpartum within 8 h.Retention of fetal membranes from 6 to 24 h post parturition is termed retained placenta.It has been published in various journals in form of reviews and researches (Biner et al., 2015;Jemal, 2016;Swiefy, 2003;Tucho and Ahmed, 2017).It is better to illustrate the solution for retained placenta in developing countries.Ethinoveterinary have showed a great role in the treatment of animal diseases in developing country.Therefore, this paper stands to illustrate and coin out the problem, and solution to ROP in ruminant animals.
ROP can create some disorders within reproductive organ of animals by allowing microorganisms to grow inside the uterus thereby causing inflammation of uterus, fever, weight loss, decreasing milk yield, longer calving intervals and if the case is severe animals may die.Moreover, the animals which are in retained placenta can suffer from tetanus caused by tetanus organism which is commonly present in feaces and in the soil, and it requires at least 1 to 3 months as long term therapy.ROP can affect the economy of developing country, by decreasing economic growth rate mainly due to decreased milk yield and calf drop (Amin et al., 2013).Although the actual causes of retained placenta are not clear; the condition usually follows dystocia, maternal hypoimmunity, mal and unbalanced nutrition, stress, hereditary predispositions or infections (Hanafi et al., 2011).

PHYSIOLOGICAL ATTACHMENT AND DETACHMENT OF FETAL MEMBRANE
Cattle have cotyledonary placentas, wherein the fetal cot yledons are attached and envelop the maternal caruncles, forming the placentome.This connection is facilitated by villi from the cotyledons, and microvilli interactions at the cotyledon-caruncle interface.Collagen links the interface together at several sites, and the breakdown of this collagen is likely a key factor in placental separation (Eiler and Hopkins, 1993;Eiler and FECTEAU, 2007) (Figure 1).
The normal sequence of events initiating parturition involves fetal cortisol induction of placental enzymes that direct steroid synthesis away from progesterone and toward estrogen.Estrogen stimulates parturition by increasing the expression of the genes associated with myometrial excitability and contraction, and increases uterine sensitivity to oxytocin during the course of pregnancy.The increased estrogen can be up regulating the oxytocin receptors on the myometrium and secretion of prostaglandin F2 alpha (PGF2α).Therefore, oxytocin and prostaglandin produce strong contraction of uterine muscle (Fuchs et al., 2001).
Prostaglandin initiates myometrial contractions and results in lysis of the corpus luteum (CL).Lysis of the CL leads to secretion of relaxin and a further decline in progesterone.Both the secretion of relaxin and the decline of progesterone promote collagenase activity.Relaxin is well known for causing collagen lysis resulting in the softening of the cervix and relaxation of the pelvic ligaments.Thus, relaxin might also promote collagen breakdown at the fetal cotyledon-maternal caruncle interface.Conversely, progesterone promotes myometrial quiescence and suppresses collagenase activity.Thus, the decline in progesterone during the prepartum period could make allowances for the enzymatic activity necessary for placental separation (Fuchs et al., 1999;Maj and Kankofer, 1997).The mechanism of placental separation is detailed in Figure 2.
In the normal situation, serotonin plays a great role in the attachment of placenta.High fetal and placental serotonin during pregnancy could help to maintain placental attachment by promoting placental cell proliferation and inhibiting matrix metalloproteinase (MMP) activity (Fecteau and Eiler, 2001).The maturation of the fetal monamine oxidase enzyme system close to parturition could results in the metabolization and subsequent decrease in serotonin, which in turn could promote placental separation and parturition (Fecteau and Eiler, 2001).In addition to changes in the hormonal environment that favors the enzymatic breakdown of cotyledon-caruncle linkages, activation of the maternal immune response against the fetal membranes can play an important role in the breakdown of the placenta.Increased leukocyte chemotaxis and activity occur in cows with normally expelled placentas, and the cytokine interleukin-8 plays the role of a neutrophil chemoattractant in the cotyledon during parturition (Kimura et al., 2002).
The maternal immunological recognition of fetal MHC Class I proteins expressed by trophoblast cells triggers an inflammatory response that contributes to trophoplacental separation.These molecules, absent in early pregnancy, are expressed by fetal trophoblast cells in the 3rd trimester of pregnancy, and could play a role in initiating an inflammatory response that ultimately dissolves the adhesions between maternal and fetal portions of the placenta (Davies et al., 2000(Davies et al., , 2004)).Trophoblast secretes interferon-tau (IFN-τ) and both trophoblast and endometrium secrete prostaglandin E and the endometrial glands secrete serpins (uterine milk proteins), all of which inhibits lymphocyte activation to keep on the embryo not rejected by the dam (Hauguel-de Mouzon and Guerre-Millo, 2006).Class I MHC antigens expressed by trophoblasts prevent maternal NKmediated cytotoxic responses (Entrican and Wheelhouse, 2006).Some researches propose that placental retention after normal parturition is more common when the presence of MHC Class I compatibility occurred between the dam and calves.This MHC Class I compatibility implies genetic similarity in the MHC locus between fetus and dam which result in deficient alloreactivity of the maternal immune system against fetal antigens.All this leads to lack of cytokine production such as interlukin-2 and tumor necrotic factor alpha that are necessary for the maturation and shedding of placenta (Davies et al., 2004;Joosten and Hensen, 1992).
The increased cortisol concentrations in cows that developed retained placenta have immunosuppressive and inhibitory effects on leukocyte migratory activity (Engler et al., 2004).High progesterone and cortisol levels in the blood in stressed cows may induce accumulation of immunosuppressive proteins in the uterine lumen; this makes the uterus susceptible to infection and persistence of bacteria (Azawi et al., 2008;Kaczmarowski et al., 2006).

THE MECHANISM OF PLACENTA RETENTION IN RUMINANT ANIMALS
Pregnancy in dairy cows is considered to induce oxidative stress, which in turn can be a significant underlying factor to dysfunction host immunity and inflammatory responses and can increase the incidence of perinatal disorders (Sordillo and Aitken, 2009).The pathogenesis pathway of retained placenta development commences with an imbalance of antioxidant or oxidant capacity, decrease in estrogen production, decreased PGF2 and accumulation of arachidonic and linoleic acids in the placental tissue.Therefore, the diminishing effect of antioxidant can cause retained placenta in ruminant animals.It is necessary for the animal organism to maintain its antioxidative/oxidative process in the balance during prenatal period (Sordillo and Aitken, 2009;Wischral et al., 2001).
When the animals approach labor, hormone like prostaglandin and oxytocin are released, resulting in mechanical contraction of the uterus that is a vital role for normal delivery.The contraction persists into stages of labor and is responsible for expulsion of fetal membrane.However, when the contraction of uterus fails, the fetal membrane stays in uterus without expulsion.Therefore, the main case for retained placenta is the failure of uterine contraction which contributes to the detachment of cotyledons from maternal caruncles, and the lack of damage to fetal villi in normally expelled membrane which is not purely mechanical (Laven and Peters, 1996).Weakness of contraction of myometrium, lesion of cotyledon, edema of villus, heparin released by mast cells and the deficiency of trace minerals are involved in the retained placenta (Hehenberger et al., 2015).In general, it is suggested that the placenta can be retained in uterus of animal due to lack of uterine contraction and inflammatory disorder which accumulate the arachidonic in placental tissue, also if the collagen which connects the caruncle to cotyledon is unable to breakdown; this causes the persistence attachment of fetal membrane in uterus of animals.
The events involved during detachment of fetal membrane or in cotyledon-caruncle detachment are listed as follows (Youngquist and Threlfall, 2006).

Morphological event
Detachment of placenta in the cow involves separation of the finger-like cotyledon villi from the caruncle crypts without significant tearing of either fetal or maternal epithelia.For the cotyledon villi to separate from the caruncle crypt, it is critical that the mouth of the cotyledon "pouch" be opened first by proteolytic enzymes.The separation of cotyledon from caruncle is achieved when the formation of the depression of the cotyledon toward the apex by digesting the edge of the cotyledon pouch occurred, then the finger-like cotyledon villi will be separated from the caruncle crypt.
After placental detachment is accomplished, uterine involution is completed in an average of 39 days in normal cows and 50 days in cows with retained placenta.By day 6 of postpartum, caruncle septa are disorganized; by day 15, caruncles are completely sloughed as a result of necrosis.Consequently, retained membranes are detached by caruncle necrosis within 6 to 10 days and not later than 17 days of post-partum.The surface of the endometrium is covered with new epithelium by day 26 to 30 post-partum (Youngquist and, Threlfall, 2006).

Biochemical events
The biochemistry of uterus after parturition is dominated by increased collagenase and protease activity that correlated with different stages of parturition, resulting in a massive breakdown of collagen and other proteins during uterine involution.

Physiological events
The physiological release of placenta is accomplished in most cows between 3 and 6 h postpartum.Cotyledon proteolysis (dehiscence) and decreasing adhesiveness (viscosity) of the cotyledon-caruncle interface fluids seem to be key factors in the release of placenta.Collagenases are capable of reducing the specific viscosity of collagen.Collagenase activity of cotyledon villi during delivery is increased in healthy cows and decreased in cows with placenta retention (Youngquist and Threlfall, 2006).
The cellular sources of collagenase and proteolytic enzymes for placental release in the cow are unknown.In laboratory animals and humans, myometrial cells, fibroblasts, and leukocytes have been identified as sources of collagenase in the uterus (Youngquist and Threlfall, 2006).
Lack of uterine motility is not considered as a reason for primary retention, since uterine motility is normal or above normal in cows with primary ROP.The direct cause of placental retention is uncertain, but it is related to a deficiency of myometrial contractions and failure of the maternal immune system to successfully degrade the placentomes at the end of pregnancy (Frazer, 2005).The pathogenesis or development of retained placenta results from failure of timely breakdown of the cotyledoncaruncle attachments after delivering of the fetus (Figure 3).The most common causes for ROP are infectious and non-infectious diseases.Abortion, stillbirth, twinning, dystocia, induction of parturition with PGF2α, metabolic disorders like milk fevers and caesarian section, can cause retention of placenta (Frazer, 2005).
The spontaneous myometrial contractility is regulated by autocrine and paracrine release of PGF2α, the disturbance of endocrine function, high progesterone and cortisol levels, and low oestradiol level traced in the blood of cows with retained placenta (Kaczmarowski et al., 2006).Hormone imbalances existing before delivery are effective in inducing ROP.Increased progesterone level in ROP may be due to failure of the placenta to produce specific steroidal enzymes that help in progesterone aromatization and its conversion to estrogens (Ball and Peters, 2004).
Progesterone, more than estrogen, inhibits uterine collagenases and slows uterine involution.Dexamethasone increases synthesis and utilization of progesterone by cotyledon tissues in the cow.These changes may contribute to blocking postpartum expressio n of cotyledon collagenases.Moreover, it has been found that glucocorticoids down-regulate collagenases (Youngquist and Threlfall, 2006).
The large granulated binucleate cells in the trophectoderm of fetal membrane play a critical role in the parturition process.Binucleate cells produce placental lactogen and pregnancy associated glycoprotein (Ward et al., 2002).The increase in cortisol causes migration of binucleate cells from the fetal side of the placenta to the maternal side.When binucleate cell is migrated and becomes degranulated, there is increase in the concentration of placenta lactogen and other pregnant specific proteins (glycoprotein) (Drillich et al., 2006).These cells fuse with the endometrial epithelium and express MHC Class I on their surface.The villous portion of the placentome is the area of tight attachment and nutrient exchange.MHC Class I expression occurs prior to binucleate cell migration in this areas (Davies et al., 2000).
The cortisol on the fetal side which comes from the fetal adrenal gland is the first critical initiation of placental separation or detachment.The fetal adrenal gland is stimulated by adrenocorticotropic hormone due to maturation of the fetal hypothalamus and stress axis near the time of expected parturition.The fetal cortisol can reach a critical level of about 30 h prior to normal parturition; the increasing cortisol concentration is only on the fetal side of the circulation.Any increase in circulating cortisol in the maternal circulation is due to production of cortisol by the dam adrenal gland (Ward et al., 2002).

General fact for treatment of retained placenta in ruminant animals
The treatment of ROP is carried out by detachment of the membrane in order to avoid or reduce the inflammation and other disease, decrease milk losses, and reduce reproductive inefficiency.The therapy treatment of retained fetal membrane is a controversial subject.Intrauterine antibiotics are routinely administered, although their effect on fertility parameters is under question (Biner et al., 2015).Retained placenta can be treated using different methods such as manual removal, administration of intrauterine antibiotics, administration of hormonal therapy, and the use of plant medicine (Ethinoveterinary methods).
The manual removal of the placenta remains a common practice in every veterinary clinic, despite many studies had failed to demonstrate its benefit on reproductive performance.A study revealed that comparing manual removal and intrauterine antibiotic therapy along systemic treatment of febrile cows found no difference in reproductive outcomes, when compared with the use of systemic therapy of the febrile cows alone (Drillich et al., 2006(Drillich et al., , 2007)).
Manual removal can result in a more frequent and severe uterine infections.More conservative treatment and manual removal prolonged the interval from calving to1 st functioning CL by 20 days.Removal of an attached placenta can cause damage to the endometrium and suppresses uterine leukocyte phagocytosis, both of which encourage bacterial invasion and they all have effect on fertility of animals (Drillich et al., 2007;Eiler and Hopkins, 1992).Tetracycline antibiotics commonly used for intrauterine treatment in cattle inhibits matrix metalloproteinase (MMPs) and interferes with the normal placental detachment mechanisms.The breakdown of collagens plays a role in placental detachment and infusion of collagenase which can be helpful in breaking the caruncle-cotyledon bond in retained fetal membrane.Injection of 1 L saline containing 200,000 IU of bacterial collagenase into the umbilical arteries of retained placentas caused earlier placental release than untreated contemporaries (Sheldon et al., 2006).
If applied within 24 to 72 h after calving, collagenase treatment can cause release of membranes in cows within 36 h.This treatment is targeted specifically at correcting the lack of cotyledon proteolysis and might be more effective than traditional therapies (Eiler and Hopkins, 1992).In general, manual removal and giving of intrauterine antimicrobial have been practiced to treat retained placenta (Sheldon et al., 2006).
Vitamin supplementation like antioxidants, vitamin E and selenium by single IM injection at week 3 prepartum is used as prophylactic dose to avoid placental retention in cows.The hormonal treatment of ROP is done by administration of prostaglandin, oxytocin and other steroidal hormones.PGF2α does not cause detachment of retained membranes, but it can improve reproductive performance in the early postpartum cow due to the uterokinetic effect (Gupta et al., 2005;Hauguel-de Mouzon and Guerre-Millo, 2006).Glucocorticoids (dexamethasone) could have a direct inhibition effect on collagenase activity which inhibits PGF2α synthesis within cotyledon cells (Gupta et al., 2005).

Abdisa 141
Oxytocin is the best hormonal choice for treatment of retained placenta in the early postpartum in animals.20 IU three to four times daily have been used for retention of placenta, which acts as uterokinetic (Youngquist and Threlfall, 2006).Antimicrobial agent is also important in such treatment and it can treat secondary bacterial infection as well.The antibiotic required in the treatment of ROP are called intrauterine antimicrobial like tetracycline (powder 5 g) and procaine penicillin G intramuscular (IM) on day 1 to 3 which is used in endometritis (Youngquist and Threlfall, 2006).The treatment of retained placenta has been carried out by many clinicians and veterinarians; they used different methods such as manual removal, hormonal and nonhormonal like intrauterine antimicrobial and the like.This paper stands to illustrate the best treatment for ROP in remote areas; places where there are no clinics or drugs available, the drug for treatment of retained placenta is prepared from natural plant species.

Plant source of medicine used for treatment of retained placenta in ruminants
Plants are sources of various chemicals and secondary metabolites which have medical value in human and animal health.In the traditional way, the farmers treat their animals by administration of plant extract juice in patient animals.The herbal or plant prescriptions for treatment of retention of placenta are characterized as antibiosis, antiphlogosis and immune enhancement and fertility improvement without endometrial injuries.The survey data collections were conducted in Oromia region.The following plants have been used for treatment of ROP in Ethiopia: Flax seed (Ija talbaa), Vernonia amygdalia (Eebicha), Dodonea angustifolia (Itacha/Hitacha), Ensete ventricosumis(Warqee), Grewa ferrugina, Solanum acaule (Iddii hoolaa), Solanum acuminatum(Iddii saree), Dovyalis spp.(Koshommii), Galinsoga quadriradiata (Waasee), Plumbago zeylanica, Momordica spp.(Qorii simbiraa), Colocasia esculenta (Goodarree), Bryophyllum pinnatu (Bosoqqee) and Urera hypselodendron (Laanqessaa) summarized in Table 1.These plants are sources of secondary chemical metabolites which act as antioxidant, antimicrobial and anti-inflammatory used to relief animal from pain and removal of placenta.In Ethiopia, especially in Oromia region, plants part like roots, leaves, stems, barks and seeds are collected and dried in order to prepare the extraction of chemical in form of powder and juice.The collected plants are used for treatment of ROP with a combination of other plants in order to increase the effectiveness of chemical.The combination of two different plants extract is better than extract chemical from a single plant.The following plants are common in the treatment of ROP.The preparation methods are like the root of Momordica spp.(Qorii simbiraa) which is

Ricinus communis Euphorbiaceae
Leaves, seed The dried part of the Ricinus communis is mixed with water and given for cow to repulsion of placenta (Birhanu et al., 2015).

Koshommii Dovyalis spp. Flacuourtiaceae Leaf
Leaf of Dovyalis is chopped and mixed with hot water and given for animal orally (Tolossa et al., 2013) Qorii simbiraa Momordica spp.

Cucurbitaceae
The root of Momordica spp is collected, dried ground and mixed with the powdered root of Colocasia esculenta, then this mixed powder is soaked in warm water and one cupful filtrate drenched into animals (Tolossa et al., 2013) Bosoqqee

Bryophyllumpinnatu Crassulaceae Leaf
The latex part of leave or stem is mixed with Urera hypselodendron (Laanqessaa) collected and applied into intrauterine of cow (Tekle, 2014).collected, dried, ground and mixed with the powdered root of Colocasia esculenta, and then this mixed powder is soaked in warm water and one cupful filtrate drenched into animals.After 24 to 48 h, the placenta would be ejected from the suffered animal.Dovyalisspp (Koshommii) is used to remove retained placenta, the leaf will be chopped and mixed with hot water and given to the animal orally (Tolossa et al., 2013).
In rural areas where modern medicine is inaccessible to farmers, Ethnoveterinary medicine (EVM) is often used to expel retained placenta in livestock.Ethnoveterinary medicine is a scientific term for traditional animal health care that encompasses the knowledge, skills, methods, practices, and beliefs about animal health care found among community members (McCorke, 1986).
Ethnoveterinary medicine is the community based local knowledge and methods in caring, healing and management of livestock in remote area where modern medicine is limited, and it has been developed through some trial and errors, and experiments (Misra and Kumar, 2004).Ethnoveterinary medicine is sustainable and ecologically sound because plant products with recognized medicinal properties are far more accessible to the villagers than Western medicine.The EVM is a cheap and easy accessible alternative to expensive pharmaceuticals, this help the farmer make it available at low cost or no cost at all (McGaw and Eloff, 2008).The traditional remedies which have been used in treatment of retained placenta throughout the world are like salty water, soap detergent solution, Terminalia serecea roots, Spirostachys africanum bark and Burkeaa fricanum bark.Other herbal plants included Ziziphus mucronata, Peltophorum africanum, Elephantorrhiza elephantina, Pouzolzia mixta, Dicerocaryu meriocarpum, Asparagaus spp., Hermaniaguerkeana, Ozoroapaniculosa, Scacdoxus spp.and Boscia albitrunca (Moreki et al., 2012).The remedies are administered either as a decoction or infusion.In the present study, the common plant parts used for treatment of retained placenta are barks, roots and bulbs and parts and products of animals such as skins and hides, bones, milk, butter and even urine and dung, are ingredients of Ethinoveterinary medicine (Balaji and Chakravarthi, 2010).

Methods of preparation and administration of medical plant for animals
The preparations of these plants are, first collection of the leaves, roots and barks of the plant, then extraction of chemical in form of juice and powder, after which they are mixed with each other.After the plant extraction is prepared, the extraction is administered into the animals orally or through vaginal application of animals; this is done to detach the fetal membrane from its attachment.As the data were collected from community, they are informed that the methods of drug preparation from plants are concluded as follow; first the collection and identification of the plant, then drying the plant part like leaf, stem and root by putting it under shade and making it into powder.Afterward it is mixed with water and if available with salt water, then administered to animals through oral and intrauterine.The retained placenta would be removed from animal after 2 to 3 days.The most common plant which have been in use in Oromia region is flax seed (Linum usitatissimum).This flax seed is first grinded to form powder, then mixed with water and if available salt water( Na cl).After everything is prepared from powder of flax seed directly administering into animal orally.After one day the placenta will be removed from dairy cow and ewes.The common plant parts which are used in preparation of drug are leaves, stems, seeds, roots, barks and cell wall combs.

Vernonia amygdalia
Vernonia amygdalia, member of Asteraceae family, is a small shrub that grows in tropical Africa.V. amygdalia is a rapid regenerating soft wooded shrub of 2 to 10 m tall with petiolate leaves of around 6 mm in diameter.V. amygdalia is valuable medical plant that is distributed throughout Africa (Ijeh and Ejike, 2011;Yeap et al., 2010).It is used as active anti-cancer, antibacterial, antimalarial and antiparasitic agent (Clement et al., 2014;Yeap et al., 2010).As one study revealed, the leaf extracts of V. amygdalia have hypoglycaemic and hypolipidaemic properties in experimental animals and is used in managing diabetes mellitus (Iwara et al., 2015).
The common name of this plant varies in ethnic groups around world.In Ethiopia it is termed as "Eebicha" in Afaan Oromo, in Amharic it is termed as "Grawa'.The methods of chemical extraction from leaves, stem and bark of V. amygdalia has been reported in many researches.In the traditional way, the community prepares the extraction by grinding and filtering the leaves of the plant.
First step is collection of the plant parts, then keeping it dry and after that, they make powder and mix with water, filtering with some filter paper or piece of cloth and administering to the animals.In the modern ways, first collection of the plant is submitted to the laboratory and the chemical is extracted using different procedure of chemical extraction (Odey et al., 2012).
The leaves of V. amygdalia has been used to treat retained placenta by mixing with table salt, then administered to cow orally, and after some time the animal is relieved from retained placenta (Birhanu and Abera, 2015;Tekle, 2014).The ethanolic leave extract of V. amygdalia have secondary metabolity chemical which acts as antimicrobial such as terpenes, oxalate, coumarins, lignins, sesquiterpenes, phytate, tannins, saponins, flavonoid, cyanogenic glycoside, alkaloids, anthraquinon,steroid and phenol (Owoeye et al.,2010;Udochukwu et al., 2015 ).The antioxidant, antimicrobial, anti inflamatory, antiseptic and disinfectant, antifungal, anticancer and antifouling effect of the chemical extracted from V. amygdalia is used to relief the animals from retained placenta and other complicated dis eases (Igwe et al., 2015).
There are so many plants which are important in the treatment of retained placenta in domestic animals and even in human beings.The chemical which extract from those plants act as antioxidant, antimicrobial and initiates the contractility of the uterus, thus placenta are easily detached and removed out of the uterus.It is suggested that the tannin produced from the plant act as astringent to facilitate the contractility of uterus and removal of placenta.
When the wall of the uterus undergoes contraction, there may be shrinkage of small blood vessels and villi and the fetal caruncle easily detached from cotyledon of maternal, as a result, expulsion of placenta may occurred.Tannins containing plants are effective where protection of underlying tissues by skin or mucosa is compromised as seen in the case of wounds and retained placenta.Tannins proteins complexion limits fluid loss and forms a physical barriers to further tissue insult (Bruneton, 1995).Astringent property of tannins has a physiological effect such as contraction of the uterus which might facilitate the removal of retained placenta.By shrinking of the small blood vessels, the capillary pressure is lessened and separation of fetal membranes occur (Bruneton, 1995;Manspeaker, 2007).Ricinus communis has been used to treat retained fetal membrane (Birhanu and Abera, 2015).In treating retained fetal membrane, the leaf of Ensete ventricosum is also given for cow (Mesfin et al., 2016).
The plant extract of Grewia ferrugina were useful for easy expulsion of placental membranes.The feeding of bamboo leaves (Bambusa vulgaris) mixed with black pepper (Qunda barbaree) helps in expulsion of placenta.Raspberry leaves when fed to pregnant mares during the last 45 days of gestation reduce the incidence of periparturient diseases viz prolonged labor, retained placenta (www.hillagric.ac.in).
Argemone Mexicana (Kossorruuadii) is used to remove retained placenta.100 g whole Argemone mexicana plant with any available local grass for feeding once a day is used for removal of retained placenta in cows (Yadav et al., 2014).
Opuntieficus indica (Adamii ishee battee) and Urera hypselodendron is used for expulsion of placenta retention by chopping the leaves and mixing with water and administering to the animal orally (Bobaso, 2016;Tekle, 2014).The chemical extraction is being performed in laboratory by different methods.However the modern extractions of chemical from plant parts are not known at the remote area where modern laboratory is scant.The farmers were used to administer the traditional medicine without any extraction, this paper stand to report how to treat the retained placenta and identification of which species of plant are used for treatment of retained placenta in Ethiopia.Generally in Ethiopia, so many medical plant species exist.Out of these plants, the most common medical plants are rich in secondary chemical metabolites, which act as antimicrobial, antiparasitic, antispasmodic, analgesic, anti-cancer and anti-fungal and etc.The discussed plants are not published in any journal, this paper is pioneer to this study, and the most common way in treatment of ROP by chemical extraction is from the mixed chemical of different plant in order to increase the broad spectrum effect of drug.
The broad spectrum effect of this extracted drug acts a s antiflamatory, antispasmodic, antioxidant and antimicrobial.The extraction from the mentioned plants also used in enhancing the production of estrogen, and increasing contraction of uterus which facilitate removal of retained fetal membrane.Actually, the community used to treat their animals by separating plant extraction, and mixing the extracted chemical with other different chemicals.
All plants are effective, but drug is more effective when extractions of different plants are mixed rather than single plant.The orally drenched is more effective than others, because the toxicity of this extracted chemical is biotransformed and absorbs into the system, thus it easily reaches the uterus wall.The intrauterine application is also effective, however it is not practiced much in community.Moreover, the intrauterine infusion of this extracted drug from the mentioned plants may causes some disorder, and the toxicity of that chemical may have effect on the fertility of animals.Therefore, from this research it can be said that the orally drenched of extracted chemical from Ethinoveterinary medicinal plant is better than intrauterine infusion in the treatment of ROP.

Conclusion
Retained placenta is common in domestic animals like ruminants and equines.The most common causes of ROP are dystocia, caesarian section, maternal hypoimmunity, mal and unbalanced nutrition, stress, hereditary predispositions or infections, failure of uterine contraction.Retention of placenta in ruminant animals have negative influence on the economic development of developing country by decreasing milk production, the inflammation of uterus, fever, weight loss, longer calving intervals and dying of animals.The treatments for retained placenta have been operated by different methods like hormonal and non-hormonal methods.The plant medicine which is used for treatment of retained placenta in ruminant animals are listed as follow:  (Laanqessaa).The effects of retained placenta in domestic animals are critical in increasing mortality rate and decrease of economy in developing country.Therefore, this paper leads and gives the solution for the treatment of ROP in developing countries or remote areas where modern medicine is not available and veterinary clinic is scant.This paper also tends to apply some concept and information for production of drug from the mentioned plants in future industry.

INTRODUCTION
Domestic animals in Ethiopia are mainly used as drought animals, source of milk, meat, hide and skin and as pack animals.Animal diseases are one of the primary constraints in increasing the productivity of food animals in sub-Saharan Africa (Lemma et al., 2001).
Lopez (2012) stated diseases affecting the respiratory system are generally the leading causes of morbidity and mortality in large domestic animals.The management and environmental stress are often the decisive factors for the development of clinical diseases (Howard, 1993).
Environmental and management stresses like over work, undue exposure to cold winds and rain, sudden change of climate, insufficient food and chronic diseases particularly trypanosomosis are responsible for respiratory diseases (Andrews and Kennedy, 1997;Dungworth, 1993).Pulmonary tissues are damaged by potential bacterial pathogens especially Pastuerella infection that normally reside in the upper respiratory tract (Carter, 1984).Even the lung tissues can be found infected with food borne parasitic zoonotic infections *Corresponding author.E-mail: kiduney06@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License (Sandhu et al., 1995;Chhabra and Singla 2009).
The reaction of the lung tissue may be in the form of an acute fibrinous process as in pasteurellosis, a necrotizing lesion as infection with Fusobacterium necrophonrum or as a more chronic caseous or granulomatous lesion in Mycobacterial or Mycotic infections (Radostits et al., 2007).
The gross and histopathological characterization of the pulmonary disease has not yet been studied in Mekelle and around slaughter at Abergelle Export Abattoir; for this reason this study is designed with the objective to assess the major pulmonary lesion of the slaughter cattle based on lobular distribution of the infected lung and to identify as well as characterize the types of gross and histopathological lesions in lungs of cattle slaughtered at Abergelle Export Abattoir.

Study area
The study was conducted in Abergelle Export Abattoir, Mekelle.Mekelle is the capital city of Tigray Region.It is located around 780 km north of the Ethiopian capital City Addis Ababa, at a latitude and longitude of 13°29′N 39°28′E, with an elevation of 2084 m above sea level.The mean annual rainfall of the area is 628.8 mm.The annual minimum and maximum temperature is 11.8 and 29.94°C, respectively (RSTBARD, 2009).

Study type and study animals
A cross-sectional study was undertaken to establish the prevalence of gross and histopathological pulmonary lesions of cattle slaughtered at Abergelle Export Abattoir, Mekelle.The study was conducted purposively on 240 cattle subjected to slaughter from different parts of Tigray region.All cattle examined in this study were male greater than six years of age and have good body condition were apparently healthy during ante-mortem inspection.

Sample collection and processing
Immediately after slaughter, post-mortem examination was made by visual examination, palpation and incision for the presence of any lung lesion.The gross appearance, location as well as size of the lesions and observations made on incision were also recorded .Out of 240 cattle lungs examined, a total of 208 lung tissue samples with gross pulmonary lesions were collected for histopathological study.Tissue samples having a thickness of up to 2 to 3 cm were collected from every pulmonary lesion.They were taken from the margin of healthy and affected tissue parts and fixed in 10% buffered neutral formalin (BNF) immediately.Each sample was fixed separately and transported to pathology laboratory for processing.After fixing tissue samples for 24 h, all samples collected for histopathological study were dehydrated in alcohol, embedded in paraffin, sectioned at 4 to 6 µm thickness and stained with Haematoxyline -Eosin stain.The stained samples were examined using light microscope.Sample collection and processing was done as per the method described by Assegedech (2005) and Bancroft et al. (1996).

Data analysis
The data was entered into Microsoft excel spread sheet and coded Yalew et al. 149 appropriately.For data analysis, SPSS version 15 was used.In this data analysis, descriptive statistic was used to determine the proportion of the different respiratory lesions and their lobular distributions.Chi-square was used to test the lobular distributions of pulmonary lesions.

Pulmonary lesions
Out of the total 240 slaughtered cattle examined during the study, 208 cattle (86.6%) were found to have pulmonary lesions.On the basis of gross and microscopic examination pneumonia, pulmonary emphysema, pulmonary congestion and oedema, hydatidosis, pulmonary abscesses and atlectiasis were identified with prevalence of 33.33% (n=80), 31.25%(n=75), 12.08% (n=29), 6.67% (n=16), 2.08% (n=5) and 1.25% (n=3), respectively.The frequency and prevalence of pulmonary lesions identified in this study was summarized in Table 1.The lobular distribution of pneumonia, pulmonary emphysema, and pulmonary congestion and edema were significantly (p-value = 0.000) higher in the right cranial lobe than the other lobes of lungs.However, pulmonary abscess was significantly (p-value = 0.000) higher in the right middle and caudal lobes of the lungs compared to the other lung lobes.Hydatidosis, was also most frequently encountered in the right caudal lobe (p-value = 0.000).In addition, atelectasis was most commonly encountered in the caudal lobes of both right and left lungs with p-value < 0.321.The lobular distributions of pulmonary lesions were summarized in Table 2. Representative images of some pulmonary lesions are indicated in Figure 1.

DISCUSSION
Out of 240 cattle lungs examined, 86.6 % had pulmonary lesions.This result was in agreement with the report of Gebrehiwot et al. (2015) and Abayneh (1999) who reported pulmonary lesions of cattle with prevalence of 86.2 and 83.87%, respectively.However, the result of this study was higher than the findings of Amene et al. (2012) and Ahmed et al. (2013) who reported pulmonary lesions with prevalence of 46.22 and 44.6%, respectively.These all results showed that respiratory diseases are highly prevalent in cattle production area.This high prevalence of pulmonary lesions could be due to the older age of the animals at slaughter with possibility of exposure to one of the agents which causes respiratory disease through time at least once.
The overall prevalence of pneumonia in this study was 33.33%.This agree with the report of Ahmed et al. (2013) who reported pneumonia in cattle at a prevalence of 28.7% but higher than the results of Yifat (2011) and Amene et al. ( 2012) who reported 1.8 and 1.11%  prevalence of pneumonia in cattle, respectively.The factors affecting are different in management systems, breed of animals, nutrition, climatic factors, and animal health extension services etc. Emphysema was found to be the second most prevalent lesion with a prevalence of 31.25%.This result coincides with the report of Gebrehiwot et al. (2015) where emphysema was observed on 36.3% of cattle and higher than the studies of Abayneh (1999) and Amene et al. (2012) who reported emphysema at prevalence of 16.53 and 6.77%, respectively.The emphysema could be due to excessive destruction of alveolar walls as a result of an imbalance between proteases produced by phagocytes and antiproteases produced in the lungs as a defense mechanism, secondary to obstruction of outflow of air or agonal at slaughter as indicated by Lopez (2012) and Dungworth (1993).Emphysema was encountered more frequently on the right cranial lobes of the lung.
Pulmonary oedema and congestion occurred at a prevalence of 12.08% and this was higher than the results of Amene et al. (2012) and Ahmed et al. (2013) who reported a prevalence of 2.33 and 6.5%, respectively.However, the result of this study was lower than the results of Rahman et al. (2003) and Gebrehiwot et al. (2015), who reported pulmonary oedema and congestion in cattle at a prevalence of 61.53 and 38.5%, respectively.Pulmonary oedema and congestion provides an ideal environment for the growth of pathogens of relatively low virulence.
Hydatidosis was encountered in 6.67% of the examined cattle lungs and lower than the findings of Rahman et al. (2003), Gebrehiwot et al. (2015) and Amene et al. (2012) who reported prevalence of 25, 18.3 and 35.88%, respectively.This difference in the prevalence of hydatidosis may be due to variation of dogs and wild carnivore population.Hydatid cycts were distributed most frequently on the right caudal lobes of the lungs.This could be due to the larger size of the caudal lobes, which in effect get a greater volume of blood supply (Lopez, 2012).
Pulmonary abscesses were encountered in 2.08% of the cases.This result was higher than the result of Amene et al. (2012) (Lopez, 2012;Dungworth, 1993).Additionally the two less common causes of pulmonary abscess are aspiration of foreign bodies and direct traumatic penetration of the lungs (Lopez, 2012;Dungworth, 1993).
The prevalence of atelectasis in this study was 1.25%.
Atelectasis is common when collateral ventilation is less (Lopez, 2012).The cause of usual atelectasis is occlusion of the bronchus or bronchiole, which supplies it.This results most often from a plug of mucus or purulent exudates.The air contained at the time the bronchus is closed is absorbed in a short time as is regularly the case with entrapped gases.The airless alveoli then collapse under surrounding pressures (Lopez, 2012;Jone and Hunt, 1983).It also accompanies space occupying lesions in the thoracic cavity or on the lung parenchyma like neoplasia, granuloma, hydatid cyst etc and the accumulation of transudate and exudates when their volume is large (Lopez, 2012;Jone and Hunt, 1983;Dungworth, 1993).In all types of atelectasis the collapsed lung is prone to secondary infections (Lopez, 2012).

CONCLUSION AND RECOMMENDATIONS
Out of the total 240 lungs of cattle examined, 86.6% were found with one or more pulmonary lesions, indicating pulmonary diseases as an important constraint of cattle production.The lesions encountered in this study will also play a role as a predisposing factors for respiratory disease outbreak under the influence of stress factors such as environmental change, extremes of climatic conditions, transportation and shortage of feeds and water or alone.Therefore, the prevailing environmental condition undue exposure to cold winds and rain, sudden change of climate coupled with the management stresses like over work might reverse these hidden inactive lesions and thereby contribute for the higher occurrence of respiratory diseases in cattle's.
Taking these facts into consideration the following recommendations are forwarded: 1. Further and subsequent study is recommended on a larger scale with specific data about the origin of animals slaughtered.2. Awareness of the public about hydatidosis should be increased through public education and infected organs should be properly disposed.

3.Strong collaboration among governmental organization, nongovernmental organization, veterinarian (researchers)
and livestock owners should be made in order to determine the impact of respiratory disease on cattle production and to design control and prevention strategies.

Bovine coccidiosis is a protozoan infection caused by different species of
Eimeria which has a worldwide distribution.The disease which mainly affects calves belongs to large herd size where hygiene is not well managed and is associated with poor body condition.A cross-sectional study was conducted from November 2016 to April 2017 to determine the prevalence of bovine coccidiosis and identify the associated risk factors in semi intensive and extensive dairy farms in and around Holeta town, Finfine Zuria Liyu Zone, Oromia Regional State, Ethiopia.Fecal samples were randomly collected from three hundred and eighty four calves belonging to dairy farms and examined for the presence of the oocyts of Eimeria by floatation technique using saline solution.The study revealed that the overall prevalence of coccidiosis was 26.04%.The risk factors considered were age, sex, breed, production system, herd size, fecal consistency, body condition and hygienic status of the house.The prevalence of coccidiosis was higher within calves in poor hygienic (58.6%) dairy farms than calves from better hygienic farms (14.6%).There was also significant difference (P<0.05) in the prevalence of coccidiosis between different herd sizes with higher prevalence in herd size >10 animals (39.3%).The highest prevalence of coccidiosis was recorded in calves with diarrheic faeces (91.7%) than calves with soft, constipated and normal fecal consistency (P<0.05).Appropriate monitoring and control of the disease is advisable in the study farms.Key words: Calf, coccidiosis, dairy farms, Eimeria, Holeta, prevalence.

INTRODUCTION
Ethiopia is endowed with abundant livestock resources of varied and diversified genetic roles with specific adaption to its wide range of agro ecologies (CSA, 2015).This great livestock potential is not properly exploited due to many prevailing socio economic values and attitudes, traditional management methods, limited genetic potential and rampant disease.
Gastrointestinal parasite infections are a problem for both small and large scale farms; however, their impact is greater in sub-Saharan Africa.The prevalence of *Corresponding author.E-mail: Temesgen.kassa@yahoo.com.Tel: +251933864380.Fax: 0112370377.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License gastrointestinal parasites and the severity of infection vary considerably depending on the genera of helminth and protozoan parasites involved, animal species, local environmental conditions such as humidity, temperature, rainfall, vegetation and management practices (Debela, 2002;Sandhu and Singla, 2005;Wadhawa et al., 2011 ).
The most important disease problems in the young calf are pneumonia and diarrhea.The pathogens associated with calf diarrhea are rotavirus, corona virus, Salmonella species, protozoan parasites, Eimeria and Cryptosporidium species (Bhat et al., 2012;Brar et al., 2017).Bovine coccidiosis is a protozoan disease of the intestinal tract caused by microscopic organisms called coccidia; and is one of the most common and important disease of cattle worldwide (The Merck Veterinary Manual, 2005).This disease is usually the most known and devastating protozoan disease in calves under age of one year (Ernst et al., 1987).
Previous works which were conducted in different parts of Ethiopia showed that coccidiosis is a paramount important protozoan disease in younger calves (<12 months) which were kept in poor hygienic status as well as improperly nourished with colostrum (Abebe et al., 2008;Mehreteab et al., 2012;Alemayehu et al., 2013;Temesgen, 2016).Although, coccidiosis is an important cause of calf morbidity and mortality in Ethiopia in general, and in the study area in particular, there is no previous detail information on its prevalence coccidiosis in the study area.
Therefore, this study was conducted to determine the prevalence and associated risk factors of calf coccidiosis in farms in and around Holeta, Finfine Zuria Liyu zone, Ethiopia.

Study area
The present study was conducted in extensive and semi intensive farms found in and around Holeta town located in Finfine Zuria Liyu Zone, Oromia Regional State, Ethiopia during the period between November 2016 and April 2017 to determine calf coccidiosis and its putative risk factors.Holeta is located 45 km west of Addis Ababa at altitude 2400 m above sea level.It is geographically located between 9° 3´N latitude and 38° 30´ E longitudes.The area experienced bimodal rainfall pattern with a short rainy season from February to April and the long rainy season from the middle of June to end of September.The remaining months are dry periods.The area gets an annual rain fall of 1000 to 1100 mm and the annual temperature ranges between 18 and 24°C.The total cattle population of the study area is estimated to be 175,741, out of which 172,769 (98.3%) heads of cattle are local breeds and 2972 (1.7%) are crosses kept under extensive and semi intensive management systems (WoWAHA, 2015).

Study animals
The study was carried out on 384 calves within the age of 1 month to 1 year old.The samples were randomly collected from calves reared under semi intensive and extensive management systems.Examined calves were categorized based on their age and grouped into three as: group I (1 to 4 months age), group II (5 to 8 months) and group III (9 to 12 months of age), based on house hygiene grouped into three (good, moderate and poor) and also based on size of herd grouped into three (<10, 11-20 and >20 head of calves).

Study design and sample size determination
The type of study was cross sectional with simple random sampling technique conducted between November 2016 and April 2017 to determine the prevalence and associated risk factors of calf coccidiosis in and around Holeta town.The desired sample size for this study was determined by using the single population proportion formula according to Thrusfield (2005).Since there is no previous report on calf coccidiosis in the study area, the sample size was established based on the 50% expected prevalence, 5% desire absolute precision and 95% confidence level (Cl).
Thus, the desire sample size for Pex = 0.5 is n= 384 calves included in this study.While collecting faecal samples, data related to age, sex, bred, herd size management system, body condition, fecal consistency and hygienic status of barn were properly recorded.

Faecal sample collection and examination
A total of 384 faecal samples were collected directly from the rectum of each sampled animal with strict sanitation, and placed in air and water tight sample vials.After collection, the samples were transported in ice box to Holeta Agricultural Research Center (HARC) Parasitological Laboratory for fecal examination using simple floatation technique with saline solution (Yu et al., 2011;Gupta and Singla 2012).

Data management and analysis
All data collected were entered and managed in MS-Excel software program and analyzed using SPSS 20.0 statistical software version.Descriptive statistics such as percentage was used to approximate the prevalence of calf coccidiosis in the study area.Prevalence was calculated as number of positive calves harboring Eimeria oocytes divided by the total calves examined.Chi-square (χ 2 ) statistics were used to test the association between variables.At p <0.05 was taken as statistically significant.

Overall prevalence
In the present study, out of total 384 samples tested, 100 (26.04%) were positive for the presence of Eimeria oocyts.
The prevalence was higher in Barfata (37.5%) as compared to other study localities (p <0.05) as shown in Table 1.

DISCUSSION
Calf coccidiosis causes a significant economic loss through morbidity and mortality worldwide.The result of the present study conducted in three localities of Holota town and its environs showed an overall prevalence of 26.04% coccidian infection in calves.The 26.04% prevalence of coccidiosis in this study is in line with previous studies of Bekele et al. (2012) [22.7%] in Dire Dawa, Temesgen (2016) [24.3%] in South Wollo Zone Amhara region, Ethiopia, Toaleb et al. (2011) [24.2%] in Egypt.However, the prevalence result of the present study is much lower than many of the previous reports in Ethiopia, namely, 68.1% in Addis Ababa and Debre Zeit Dairy Farms (Abebe et al., 2008), 31.9% in Kombolcha, south Wollo (Alemayehu et al., 2013), 62.5% in Asella town (Ibrahim, 2016) and 38.9% in and around Asella town dairy farms (Tsegaye, 2016).The findings of this study are also much lower than that of other countries of the world.For instance, a prevalence of 47.1% was reported in China (Dong et al., 2012).The results of the present study, however, is higher than previous reports by Gillhuber et al. (2014) (13.3%) in Southern Germany, Hussin (2016) (9.5%) in Iraq and Das et al. (2015) (11.9%) in India.Such inconsistency in the prevalence rate of coccidiosis may be due to the variation in diagnostic tests, age of the animals, susceptibility of different breeds to the disease, stress level, handling, climatic and other factors of agro-ecology, variation in the study season, number and target group of the study animals and husbandry practices (Radostitis et al., 2007a;Abebe et al., 2008;Heidari et al., 2014).
This study showed variation in the prevalence of calf coccidiosis between different study localities (18% to 37.5%).Similar results were obtained in different parts of Zimbabwe (17.4 to 32.6%) (Pfukenyi et al., 2007), Al-Baha, Saudi Arabia (29.84 to 32.51%) (Ibrahim et al., 2015) and in Addis Aababa and DebreZeit, Ethiopia (57.2 to 76.4%) (Abebe et al., 2008).This geographical difference in distribution of positive cases could be explained by the management practices and the biosecurity followed by the farm owners.
Analysis of risk factor with regard to the age of the calves revealed that there is no statistically significant association (χ2= 3.363, P= 0.186) between the age of the calves and coccidian infection.This result agrees with the reports of Abebe et al. (2008), Tsegaye (2016), Alemayehu et al. (2013), Bekele et al. (2012) and Gillhuber et al. (2014).This similarity could be due to the fact that animal husbandry practices in the study areas are identical, and also, different age groups were kept and housed together without separation.
There was no statistically significant difference in prevalence of coccidian infection between male and female animals (χ2 = 0.437, p =0.508), which is in agreement with the reports of Abebe et al. (2008), Heidari and Gharekhani (2014), Alemayehu et al. (2013) and Ibrahim (2016).This might be associated with the fact that different sex groups kept in similar husbandry system might have equal chance of accessing the oocyts.Despite this, previous studies done on adult cattle showed higher prevalence of Eimeria in female animals than in males (Manya et al., 2008;Rehman et al., 2011).Nevertheless, this could be attributed to the physiological stress loaded on female animals in relation to pregnancies and giving birth as compared to males (Radostits et al., 2007a).
The breed related prevalence of coccidian infection in the present study showed no statistical significant difference between breeds (χ2 = 0.103, p =0.748), which is in agreement with the findings of Abebe et al. (2008) and Alemayehu et al. (2013).
The possible explanation for this similarity could be the fact that calf rearing condition in the study areas was identical and different breeds were housed together without separation.In contrast to the current findings, susceptibility differences were reported between local and cross breeds (Ibrahim, 2016).This breed susceptibility difference could be related to the dose of oocytes ingested and the species of Eimeria involved in the infection (Taylor et al., 2007).
In the current study, coccidian infection was significantly higher in calves reared in large herd size than in small size (χ2 = 16.387,P = 0.000).This finding is in line with the report of Nasir et al. (2009).This similarity might be due to rapid spread of infection from calf to calf as well as greater contamination of feeding and watering troughs when animals are communally feed and overcrowded (Radostitis et al., 2007a).However, the current finding disagrees with that of Abebe et al. (2008).This variation might be due to the differences in the study seasons, husbandry practices and the treatment regime given to the calves.
There was no significant difference (χ2 = 0.281, p = 0.596) in the prevalence of coccidiosis and management systems.This result agrees with the report of Abebe et al. (2008), Alemayehu et al. (2013) and Temesgen (2016).This similarity might be due to equal chance of accessing the oocysts when grazing from contaminated field.However, the current finding is in contrast with the previous report of Abisola (2004).This variation might be due to hygienic condition of the barn, nutritional status of the calves, contamination level of the feed, water, floor and treatment given to the animals.
There was statistically significant (χ2=44.668,P=0.000) difference in prevalence rate between fecal consistency and coccidian infection which agrees with the findings of Pundit ( 2009) and Alemayehu et al. (2013).However, this finding disagrees with the report of Abebe et al. (2008).In the present study, 91.7% diarrheic calves were found tobe positive to Eimeria.However, there were no apparent clinical signs in most of the animals sampled for the study.
A strong significant association (χ2= 51.447, P= 0.000) was recorded between body condition score and coccidian infection in the current study.Similarly, Mehreteab et al. (2012) reported a higher infection rate in calves with poor body condition score than in calves with good and moderate body condition score.This might be due to the weak immune status of the calves with poor body condition score.As a result, malnutrition and other parasitic infections result in immune compromised calves.This condition produces a higher infection rate in poor state animals than in good-state animals (Radostits et al., 2007b).
In the current study, association of Eimeria infection in relation to the hygienic status of calf house was verified (p < 0.05).This result agrees with the report of Bekele et al. (2012) Mundt et al. (2005a, b) and Dawid et al. (2012).The similarity implies that poor sanitation in the calving and calf housing areas as well as poor management of housing favors infection of coccidiosis.Obviously, poor ventilation, droughts, poor calf nutrition, group pens, heavy stocking, cows present with calves, soiled bedding are regarded as risk factors for coccidiosis (Radostits et al., 2007b).

CONCLUSION AND RECOMMENDATION
In conclusion, this study provides proof of a coccidian infection in dairy farms in Holeta and its environs.Hygiene of calf's house can be considered as a risk factor for the occurrence of coccidia.Large herd size, diarrheic faeces and poor body condition of the calves also increased the risk of infection with coccidia.However, sex, age, breed and management system of calves did not show any difference with the occurrence of protozoan parasite infection.In general, different risk factors were considered to affect the rate of infection of calves with this protozoan parasite.
Based on these findings, it was recommended that calves with severe diarrhea be isolated and treated with appropriate drugs; any possibility of fecal contamination of the farm and the calves be minimized.Further epidemiological investigations are required to determine the protozoan parasite species composition and their economic impact.

INTRODUCTION
Muscle is a tissue that consists predominantly of contractile cells of animals.It has different spatial arrangements of myofibrils.The spatial arrangement of the fibrilar network of muscle has great role in the quality of the meats (Bowker et al., 2014).The pH of muscle is approximated from 6.9 to 7.0.The variation in pH may come from physical exercise, nutritional deficiencies, age, sex and/or other factors of the animals (Zhang et al., 2010;Simela, 2005;Simela, 2004).Muscle is converted into meat during the processes of rigor mortis.In the pre rigor stage, the muscle is converted into the stage of rigor and it becomes changed into meat.Fresh meat contains approximately 70 to 75% water, 15 to 20% protein, 5 to 10% lipids or fat and oils, 1 to 2% carbohydrates and 1% vitamins and minerals.At the rigor stage of meat, the amounts of these contents may be changed (Simela, 2005).Meat is said to be quality if it is suitable for use in specific products.The water holding capacity, the rate of decline in pH, flavor, juiciness, color, ethical quality and health quality are the quality indicators of meat.Good quality meat has a potential to have good water holding capacity, slow rate of declining pH, good appearance and *Corresponding author.E-mail: whaile947@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License fatty smell and does not have zoonotic diseases (Aaslyng, 2002).The quality of meat can be altered either by pre-slaughter conditions of the animals or by post mortem conditions of the meat (Miller, 2007).
Water holding capacity (WHC) of meat is defined as the ability of meat to retain the water when external force is exerted.Such forces can be exhaustive starvation and thirst, prolonged traveling on foot, extreme heat or windy air condition of the environment when the animals stay, dry or humid storage of meat, and the orientation of cutting, grinding, or processing of meat (Miller, 2007).The majority of water in meat is held within the structures of the muscle itself.It can be found within the myofibrils, between the myofibrils and the cell membrane (sarcolemma), between muscle cells and between muscles bundles (groups of muscle cells).Once muscle is harvested, the amount of water in meat can be changed depending on numerous factors related to the tissue itself and how the product is handled (Simela, 2005).Water held in the meat can have different names according to their location.Bound water is the water that is found within the protein of the meat.Entrapped water found within muscle structures.This water molecule may be held either by steric (space) effects and/or by attraction to the bound water but is not bound per se to protein.In early postmortem tissue, this water does not flow freely from the tissue, yet it can be removed by drying, and can be easily converted to ice during freezing.Entrapped or immobilized water is most affected by the rigor process and the conversion of muscle to meat.Upon alteration of muscle cell structure and lowering of the pH, this water can also eventually escape as wash out.This entrapped water is becoming free water when it is moved from the structure in pre rigor meat as a result of rigor mortis or post mortem effects.Free water is water whose flow from the tissue is unimpeded.Weak surface forces mainly hold this fraction of water in meat (Brondum et al., 2000).Maintaining as much of this water as possible in meat is the goal of meat handlers (Huff-Lonergan, 2005).
The pH level and the level of WHC of meat can be affected by the anti mortem events, such as lack of sufficient rest or extreme hot or extreme humid air condition of the lairage (pen), starvation and deprivation of water prior to slaughter of the animals for more than 12 h or the manner of handling like cutting of the meat during deboning and meat selling period of time, and while treating the meat to avoid meat spoilage can also affect the pH level and WHC of the meat (Lomiwes, 2008).This alteration of the pH level and WHC of the meat may come from the alteration of glycogen level of muscle of animals stressed during pre-slaughtered condition (Aaslyng, 2002;Miller, 2007).The stability of given meat can be ensured when the requirement of its water holding capacity is maintained and when there is a slow declining of its pH level.This means that the water holding capacity and the slowly declining of pH level of that meat have zoonotic, economic and nutritional importance (Simela, 2005).Meat inspectors and local abattoir administrators of Mekelle city mentioned that the butchers brought their animals to slaughter house directly from the local markets without getting pre-slaughter rest (Mekelle, 2016).Those animals might have travelled from a longer distance in starved condition and stressed by moving here and there in the market and sent directly to the slaughter house without giving sufficient rest prior to slaughter.Besides, most of those animals are poor in body condition, progressively exhausted in work and older age.The above-mentioned professionals and other meat consumers also mentioned that the meat found in the butcher houses in Mekelle city looks dry, dark in color and not attractive to consume it.However, the problem is not yet studied.Hence, the present work will deal with the following specific objectives.
(1) To determine the water holding capacity of beef sold in selected butcher shops of Mekelle (2) To establish the associations between age and preslaughter rest with pH and water holding capacity.

Study area
The study was conducted in Mekelle city, Tigray region, Northern Ethiopia.Mekelle is an urban centre and the capital of Tigray region.The city covers an area of about 53 km 2 , with an estimated population of 215,546 people.It is located 783 km North of Addis Ababa (Figure 1).Currently so many regional, national and international people are visiting the city.From small to larger scales of restaurants are emerging in this city.It has 160 legal butcher houses that are distributed throughout the city and there are also some illegal butchers whose number is not determined yet (TRHDA, 2008).

Study design
A cross-sectional study was employed for the present study.

Sample size
The sample was collected from Mekelle Municipality abattoir where the legal butcher shops send their animals for slaughter.For maximum precision, samples from 50% of the butcher shops, that is 80 samples were collected.

Sampling design and sample collection
Simple random sampling technique was employed to select 80 butcher shops from the total of 160 (Mekelle, 2014) butcher houses.The longissimus dorsi (LD) muscle was taken because of its economic importance (cheapest in value in relative to the other parts of the muscle parts), long muscle and easy to sample (Preziuso and Russo, 2004).In order to know the age of the meat, all samples were taken from Mekelle Municipality abattoir.Collection of fresh meat sample was done immediately following each slaughter by labeling each sample orderly in accordance to the code of the questionnaire.To prevent moisture loss during sample collection and preparation a tightly sealed plastic tube was used.

Questionnaire survey
Each sample was accompanied by questionnaire for characteristics of the owners' address, sex; characteristics of the slaughtered animal, the service provided, and observational characteristics of the meats.

Laboratory works
The work was conducted in the microbiology lab, college of Veterinary Medicine, Kalamino campus.The pH of the collected meat was measured using digital pH meter by probing it in to the meat for 30 to 60 s (Abraham and Kumar, 2000) and reading was recorded each sample along with its own code.Before taking the measurement it was calibrated using standard buffer solution (neutral) and it was rinsed with distilled water after taking each measurement.Firstly it was measured at 3 h period of time, and secondly, the same test was carried out at 24 h (Santos-Silva, 2001).The water holding capacity of meat was determined by two fundamentally different principles: Filter paper press method and the water drip loss method.

Filter paper press method
The external force used to drive out the water was the filter paper press method.Chromatography paper Whatman No. 40 was kept for 24 h in a dissociated 38% sulfuric acid in advance that it complies with 60% humidity and it helped to diffuse out the water freely through the paper (Kashif et al., 2014).Five grams of 24 h aged meat was homogenized on a metal plate.Out of this, 300 mg meat which was measured right after preparation was put on the Whatman paper No. 40 and then was placed between two slides on which a 100 g weight was placed on the top slide for 5 min so as to exert downward force and to release water from the meat as per the method described by Abraham and Kumar (2000).
The water released from the meat was wetting the paper and the boundary of that wetted area was demarcated using sharp pencil and was measured and reported in percentage of the ratio of the diameter of the meat to the diameter of the water wetted paper as per Mendiratta et al. (2008).

Water drip loss (Honikel bag method)
A 2.5 cm thickness slice, 2 × 5 cm width and length 24 h aged raw meat was cut parallel to the muscle fiber and hanged for 2 days in plastic sealed tube at 4°C as described by Brondum et al., (2000).The water drip out of the meat was measured and was reported in percentage of the ratio of weight of water loss to weight of meat prior to drip out the water (Diaz et al., 2010;Brondum et al., 2000;Honikel, 1998).

Data analysis
The qualitative and quantitative data were analyzed using simple descriptive and computer based statistical software (STATA 11) was used to analyze the data in order to compare means of age and pre-slaughter stress of the slaughtered animals on pH and water holding capacity of the meat.For the purpose of this study, p<0.05 was considered to be significant.

Questionnaire survey
The characteristics of the slaughtered animals had shown that the majority (65%) of the slaughtered animals were old.Only 35% of the slaughtered animals were at the stage of bull.More than half (66%) of the respondents did not have lairage for giving rest for the purchased animals before they were slaughtered.96.3% of the slaughtered animals were Zebu breed, only 3.7% of them were crossbreed.More than 78% of the carcasses were dark in color and stored in the butcher shops for more than

Water holding capacity
The mean value of the drip loss of the total meat samples was shown as 4.58 with a low standard deviation (0.95).
(Table 2).On the other hand, the filter paper press result was shown with the mean value of 49.4 with a relatively wider standard deviation (5.85) (Table 2).

pH at 3 and 24 h post-slaughter
The mean pH value of the total sampled meat measured at 3 h post-slaughter was 6.2 with low standard deviation (0.26), and reduced to the mean value of 5.9 and standard deviation of 0.11 pH level measured at 24 h post-slaughter (Table3).

Effects of age on pH, drip loss, and filter paper press measurements
The age had statistically significant association with the pH measured at 24 h post-slaughter, drip loss and filter paper press (p<0.05)(Table 4).

Association between pre-slaughter rest and pH at 3 h post-slaughter
There was statistically significant association between the pre-slaughter rest and the pH of the meats measured at 3 h post-slaughter (p<0.05)(Table 5).

DISCUSSION
In the present study, it was found that 65% of the animals slaughtered were old whereas 35% were young (Table1).This agrees with the report of Dawit et al. (2013) that64.4% of the animals slaughtered at Mekelle abattoir were old in age.According to 78% of respondents, they spent more than 24 h to sell for a total carcass (Table 1).This prolonged storage of the meat might expose the meat to excessive drip loss.This is well supported by the reports of Walukonis et al. (2002).It was claimed that meat with low glycogen level will have dark color and high water holding capacity (Miller, 2007).According to the present study, the meat from 78.3% of butcher shops in Mekelle was dark red (Table 1).This disagreed with a similar study in Texas by Miller (2007) who found 2.3% of the sampled meats were dark red.The results of mean drip loss of meat of the present study on 97.3% Zebu and 3.7% crossbreds (Table 1) was relatively higher (4.58%) (Table 2) than the findings of Waritthitham et al. (2010) reported as 3.09% on Brahman breed which were fed with unknown type feeds; similarly, Yuksel et al. (2011) reported 1.8% drip loss on Holstein Friesian breeds fed on specific feeds.on the other hand, the result of filter paper press of this study was with the mean value of 49.4% (Table 2) of dark colored meat that was agreed with the reports of Gunenc's (2007) that had shown with mean value of 48.75 of PSE meat (p<0.05), and Miller (2007) also reported similar findings of dark colored meat with higher dark firm dried meat (DFD).These indicated that the low water holding capacity of the meat could cost butchers house significantly which was also supported by Huff-Lonergan (2005).The pH at 3 h post-slaughter recorded was 6.1 to 6.6 (Table 3).This is comparable with the work of Troy (1999) who recorded 6.1 to 6.37.The higher pH might be  Gunenc (2007) stated that pre-slaughter stress has effect on PM pH 3. The preslaughter rest of the animals had strong statistically significant (p<0.05)(Table 5), association with PM pH 3 of the meat which agrees with the statement of Lyczynski et al. ( 2006) that "pre-slaughter stress of animals had statistically significant effect on the early PM pH of meat (p<0.05).This is possibly due to the accumulation of lactic acid in muscle of slaughtered animals which was formed as a result of glycolysis process formed by preslaughtering stress of slaughtered animals and it is agreed with the reports of Zhang et al. (2010).
Water holding capacity and drip loss of the meat had statistically significant association with the age of the animals (p<0.05).This statement is supported by the findings of Kashif et al. (2014) who stated that water holding capacity and drip loss varied (p<0.05)(Table 4). between three groups of 1.5 and 2.5 years, and greater than 2.5 years olds animals.These findings also revealed that water holding capacity of meat was found lower in old age of the slaughtered animals.This might be because of higher accumulation of fat and lower moisture content in old age animals comparing to young age animals which is in agreement with the suggestions of Zhang et al. (2010).

CONCLUSION AND RECOMMENDATIONS
Determination of the water holding capacity (WHC) of meat is most important that it is one of the quality indicators of meat.The WHC of the meat was found as 49.4% by filter paper press results and water drip loss was 4.58%.These results were found as having statistically significant associations with age and preslaughter rest of the animals.Old age and lack of preslaughter rest of the animals resulted in increased ultimate pH level.The prolonged storage of that meat for more than 24 h lead to the spoilage of that meat due to high amount of water in the meat which is conducive for bacterial growth and that could be source of food born diseases and/or cause for the vigorous loss of the profit of the sellers.
The following possible recommendations were forwarded: (1) Owners of butcher shops should get training on pre-

Figure 2 .
Figure 2. Mechanism of placental separation in domestic animals.

Figure 3 .
Figure 3.The components of caruncle-cotyledon linkage and mechanism of retained placenta.

Figure 1 .
Figure 1.Microscopic and gross representative images of typical pulmonary lesions.(A) Emphysema with ruptured alveolar wall and coalescence of alveolar space (arrow); (B) Gross lesion of emphysematous lungs; (C) Multiple hydatid cysts in lungs (arrows).

Table 1 .
The common plants used for treatment of retained placenta in Ethiopia, Oromia.

Local name (AfaanOromoo) Scientific name Family name Part of plant Use and route of administration
Warqee (Enset, kocho)Ensete ventricosum Musaceae Leaves Leaf of Enset is given for animals, and then the animal may relief from ROP(Mekonnen et al., 2016).

Table 1 .
Prevalence of pulmonary lesions in lungs of cattle slaughtered at Abergelle Export Abattoir, Mekelle.

Table 1 .
Prevalence of caves Coccidiosis in the study localities.

Table 2 .
Prevalence of calf coccidiosis in relation to various risk factors.

Table 1 .
Owners' response for the characteristics of the animals slaughtered and their carcasses.time before completely sold.Only 21.3% of them were light in color and sold before 24 h of storage time.

Table 2 .
Measurements of drip loss under gravity and filter paper press.

Table 3 .
Measurements pH of the sampled meat at 3 and 24 h post slaughter.

Table 4 .
Effects of age on pH at 24 h and drip loss and filter paper press measurements.

Table 5 .
Association between pre-slaughter rest of animals and pH at 3 h post-slaughter.