Comparative study on efficiency of sexed semen and conventional semen on in vivo produced bovine embryo quality and quantity of Boran and Holstein -Boran cross bred in Bishoftu, Ethiopia

The study was conducted from December 2019 to May 2020 to evaluate the efficiency of local convectional semen and imported sexed semen on in vivo produced bovine embryo quality in Boran and HB cross breed donor cows in Bishoftu, Ethiopia. In this study, it was hypothesized that the breed of donor cows and types of semen can influence the quality of in vivo produced embryo as well as the number of transferable embryos. Randomized experimental design was employed, and a total of 20 donor cows (10 Boran and 10H*B cross) were superovulated and flushed excluding non-responsive donor cows. Donor cows were randomly assigned to two groups: Group-1- five donors inseminated with sexed semen and group-2- five other cows inseminated with convectional semen from each breed. The results of study showed that the embryo recovery rate was 68% in H*B crossbreds and 53% in Boran donor cows. A total of 121 embryos were produced of which, 60 were transferable embryos (36 from H*B crossbreds and 24 from Boran), 39 UFOs (31 from H*B crossbreds and 8 from Boran) and 22 degenerated embryos (12 from HB cross and 10 from Boran). The results of this study showed that the number of transferable embryos produced from donors inseminated with sexed semen and convectional semen was nearly similar. However, the number of defective embryos tended to be higher with sexed semen and the number of unfertilized oocytes is significantly (p<0.05) higher in crossbred cows. This finding suggests that farmer can use sexed semen to get sex specific dairy calves in both cattle genotypes. LP, Texas, USA) and P value was calculated using ANOVA to find any significant relationship. Statistical significance of the study considered P value less than 0.05 as significant and P value greater than 0.05 as non-significant.


INTRODUCTION
Ethiopia holds the largest cattle population in Africa which is estimated to be about 59.5 million heads of cattle. The indigenous breeds including Boran cattle account for 98.20%, while the hybrids and pure exotic breeds represent 1.62 and 0.18% respectively (CSA, 2017). However, its contribution to the overall production has *Corresponding author. E-mail: mosisamd43@gmail.com, +251910276678.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License shown low productivity a due to their low genetic potential and poor technical knowledge on the part of dairy owners (Yehalaw et al., 2018). The low productivity of the country's livestock production system has been presumed to be due to shortage of crossbreed dairy cows and high number of indigenous breeds of low genetic potential for milk production (FAO, 2007). However, Ethiopia has large potential for dairy production development due to the country's favorable climate in the highlands which supports use of improved, high yielding dairy breeds and offers a relatively disease-free environment for livestock development (Ahmed et al., 2004). Following artificial insemination (AI), oestrous synchronization and mass artificial insemination (OSMAI) was operated as a second reproductive technology. Embryo transfer (ET) was also started in Debre_Zeit research center but the technology has not become expanded due to different constraints (Tegegne et al., 2016;Tamrat et al., 2018). With the OSMAI technology, one genetically superior crossbred cow can produce up to 32 embryos per year compared to the conventional method of breeding where the farmer has to wait for twelve months or more for a single calf that could be either male or female (Muchemi, 2011).
Following superovulation protocol (Larson et al., 2010), the number of viable embryos collected from a donor cow in one embryo collection session is an average of six, but varies considerably among different donor cows, with approximately 15% of embryo collections resulting in non-transferable embryos and a small number of donors producing 20-50 embryos . The unpredictable individual variation in the superovulation response is a major limiting factor determining the efficiency of MOET in breeding programs dependent on a number of factors related to breed donor, environment and management (Marja, 2017). The embryo transfer technology has been applied in wide area of the world because it increases the number of offspring that can be obtained from donor cow with high genetic value Bó et al., 2006). Again, the use of sexed semen has created an opportunity of getting embryos with a specific gender that would economically benefit beef and dairy industries worldwide (Sartori et al., 2004).
Studies on the efficiency of AI with sexed semen and convectional semen following super ovulation on in vivo production of embryo quality and quantity in different zebu breeds are limited in Ethiopia. Therefore, this study was conducted to evaluate the effect of convectional and sexed semen on quality of in vivo produced embryos as well as breed effect in response to types of semen in Boran and their Friesian crosses.

Experimental Animals
A total of 20 cycling donor cows (10 Boran and 10 crossbreds) were included in this study after clinical and gynaecological examination of the reproductive tracts for any abnormalities and to determine the reproductive status of individual animals using rectal palpation and ultrasonography (Tamrat et al., 2018). Only cycling (with active CL) and apparently healthy donor cows without any reproductive disorder were selected as candidates for superovulatory treatment.
All cows were managed under similar housing, feeding and health management and fed teff straw and grass hay as a basal diet that was supplemented with commercially prepared concentrate feed (mixed from wheat bran, wheat middling, corn, Noug cakes, and mineral salts) and green fodder (fresh grass, alfalfa, elephant grass). Feeding was based on the level of production, and stage of reproduction, while water was given daily ad-libtum. Animals were dewormed with broad-spectrum anthelmintic and vaccinated annually against anthrax, black leg, FMD, pasteurollosis, CBPP and LSD.
Ethical considerations in this study were taken seriously. None of the procedures involve undue stress to the experimental animals and animal handling has been with humane approach and hence it did not inflict any harm or unnecessary discomfort to the animals. All activities were carried out in accordance with the ethical guideline of the Addis Ababa University College of Veterinary Medicine and Agriculture after receiving the ethical approval under (Ref. No: VM/ERC/34/03/12/2020).

Study design
Randomized experimental design was employed to study the effects of breed and type of semen on the quality of produced bovine embryos. Experimental animals were grouped based on breed (Boran and crossbreds) and randomly assigned to the types of semen; local convectional semen with 30 x 10 6 sperm cells and imported sexed semen with 2 × 10 6 sperm cells. Before the start of the experiment, the ovaries of all animals were evaluated using a real-time B-mode ultrasound with 5 MHz linear array probe (SIUI, Altay Scientific S.P.A., Italy) and rectal palpation to check the status of CL and nature of the cervix. Animals with active CL and with medium or large, straight or not curved cervix were selected as candidate for the super ovulation treatment as kinked cervix difficult to pass.

Donor superovulatory procedure
A total of 20 donor Cows (10 Boran and 10 crossbreds) with active CL were assigned in this experiment. On day 0, CIDR (Progesterone 1.38 g, Hamilton, New Zealand) were inserted in both breed for seven days. On Day 4, cows were assigned to two different doses of FSH (Pluset®, Spain) treatment, regimen 250 IU for Boran and 700 IU for crossbreds. FSH was administered through intramuscular injection for four consecutive days in a decreasing dose regimen (Table 1) and twice per day with 12 h interval. On Day 6PGF2α (Estrumate®, France) was injected IM and CIDR was withdrawn on Day 7 during the last injection of FSH ( Figure 1).

Heat detection and AI of donor cows
After CIDR withdrawal, heat signs were closely observed and heat detector (ESTROTECT TM #U.S.pat. #6,467,430) was applied to control those animals with short duration of oestrous. Before insemination, one straw of semen from both types of semen (sexed and convectional) was thawed and checked for sperm motility. All Cows were inseminated twice with12 h interval post standing heat based on the observed heat sign to meet the ovulation time with post sperm capacitation.

Response evaluation and embryo collection
Superovulatory response was evaluated using a real-time B-mode ultrasound with 5 MHz linear array probe (SIUI, Altay Scientific S.P.A., Italy) by counting a total number of CL and unovulated follicles present on the ovaries prior to embryo flushing on day 7. The donors were injected 3 to 5 ml of lidocaine epidurally before inserting collection catheter. The embryos were collected using non-surgical technique of embryo flushing with 1000ml of commercial flushing medium (ViGRO™, Bioniche Animal Health, USA) and two-way Foley catheter (18 Fr 650 mm length; MOFA®, Canada).

Evaluation and grading of embryos
After flushing, the medium within an embryo filter was transferred to searching dish for searching embryos/UFOs under a stereomicroscope (Motis SMZ 140/143®, Roanoke, USA). Collected embryos/UFOs were transferred to four well dishes containing holding medium. The embryos were graded based on their developmental stage (from stage 1 = one cell to stage 9 = expanded hatched blastocyst) and quality (from quality 1 = excellent to quality 4 = degenerate) according to the manual of International Embryo Transfer Society (IETS, 2013).Depending on the IETS guidelines collected embryos were categorized as transferable embryos (those in 4-8 developmental stages and a quality grade of 1 and 2), degenerate and UFO.

Statistical analysis
The data were analyzed using STATA version 13 (Copyright 1985-2013 Stata Corp LP, Texas, USA) and P value was calculated using ANOVA to find any significant relationship. Statistical significance of the study considered P value less than 0.05 as significant and P value greater than 0.05 as non-significant.

Superovulatory response
An average of 8.7 and 11.1 of CL, 3.1 and 8 of unovulated follicles were counted from Boran and H*B cross donor cows respectively. A total of 121 recovered embryos/UFOs (75 from cross and 46 from Boran) were evaluated and classified. The number of CL counted from both breeds were not significantly (p > 0.05) different but the number of unovulated follicles were different (p<0.02). Over all, effects of breed on superovulatory response were summarized in Table 2 and maximum number of embryos/oocytes collected from one donor was shown in Figure 4.

Effects of breed on embryo yield and quality
There were no significant (p > 0.05) effect of breed on the number of transferable embryos and degenerated embryos but the number of unfertilized oocytes (Figure 3) across the two breed of donor cows was significantly Embryo recovery  (p ≤ 0.05) different, being higher in HB crosses. The summary of total collected embryos/oocytes is shown in Table 3.

Effects of types of semen on embryo quality
In this study there was no statistically significant (p > 0.05) difference between two types of semen used in producing transferable and non-viable embryos, even though the two types of semen were different in sperm cell concentration in addition to the sorting effect on sexed semen. However, there was a tendency (p<0.1) of sexed semen to produce higher number of defective embryos. The details of semen effect on embryo quality are shown in Table 4, and different stages and quality of embryo shown in Figures 1 and 2.

DISCUSSION
The current study was performed with the objectives of evaluating the effect of convectional and sexed semen on quality and quantity of in vivo produced embryos from two different breeds of donor cows. The superovulatory response in terms of average number of CL counted the mean number and the range of embryos/oocytes obtained from Boran and their Friesian crosses was similar with previous findings (Hasler, 2010). The variability between animals in ovulatory response to FSH-induced superovulation is mainly due to the differences in ovarian activity at the time of treatment (Rico et al., 2009). In this result, the breed variation was identified and there was a significant difference observed as high number of unovulated follicles counted in H-B crossbreds of donor cows as compared with Boran donors in this experiment. The use of sex-sorted semen in embryo production by superovulation of donor cow is an important tool for genetic improvement in dairy breeds, consequently increasing milk production. The result of this experiment showed that there was no significant (p > 0.05) difference between the two types of semen on produced embryo quality. However, several previous studies have shown that the use of sexed semen in superovulated donor   cow's results lower fertilization rate than when using nonsorted semen (Larson et al., 2007(Larson et al., , 2010Soares et al., 2011;Kaimio et al., 2013). As Schenk et al. (2009) reported, the lower fertilization rate could be attributed to the low sperm concentration and damage caused by the sorting process followed by freezing (dilution, laser exposure, process speed and centrifugation).
In the present study, the number of transferable embryos recovered after inseminating donor cows with sexed semen and convectional semen was not significantly (p > 0.05) different. This is contrary to those reported by Peippo et al. (2009) and Larson et al. (2010) that smaller proportion of transferable embryo were recovered with sex-sorted semen compared to the conventional one. However, number of transferable embryos obtained from sexed semen was higher in cross breed donor cows. This may show that the sexed semen used in current study is of good quality and the time of insemination was later as compared to that of convectional semen. In addition, insemination with sexsorted sperm should be conducted closer to the time of ovulation because sex-sorted sperm has a reduced life span in the female reproductive tract, which is due to possible precapacitation of the sex-sorted spermatozoa (Maxwell et al., 2004;Peippo et al., 2009).
In the current study, the number of unfertilized oocytes and number of degenerated embryos recovered from both types of semen (sexed and convectional) were not significantly (p > 0.05) different. However, the number of degenerated embryos tended to be higher (p<0.1) with sexed semen. There was no significant difference in the number of quality grade 1 but the number of quality grade 2 was higher with sexed semen. Some studies report a decrease in the number of grade1 embryos when the donors were inseminated with sexed semen; whereas others have not found any significant differences. However, Peippo et al. (2009) report that there was no effect of sexed semen on the number of grade1 embryos among transferable embryos in superovulated donors inseminated with sexed semen. The mean number of transferable embryos that recovered from cow inseminated with sexed semen was 3 which is in the range of previous findings of 2.1 and 3.8 transferable embryos per recovery reported by Larson et al. (2010) and Peippo et al. (2009) respectively.

Conclusion
The study shows that the number of transferable embryos produced from donors inseminated with sexed semen and convectional semen was nearly similar. However, high number of defective embryos tended to be higher with sexed semen and the number of unfertilized oocytes is higher in crossbred cows. This study suggests that farmers can use sexed semen to get sex specific dairy calves in both breeds of cows.