Domestic sheep in Kazakhstan may provide an interesting source of genetic variability due to their proximity to the center of domestication and the Silk Route. Additionally, those breeds have never been compared to New World sheep populations. This report compares genetic diversity among five Kazakhstan (KZ) and 13 United States (US) sheep breeds (N = 442) using 25 microsatellite markers from the FAO panel. The KZ breeds had observed and expected measures of heterozygosity greater than 0.60 and an average number of alleles per locus of 7.8. In contrast, US sheep breeds had observed heterozygosity ranged from 0.37 to 0.62 and had an average number of alleles of 5.7. A Bayesian analysis indicated there were two primary populations (K = 2). Surprisingly, the US breeds were near evenly split between the two clusters, while all of the KZ breeds were placed in one of the two clusters. Pooling breeds within country of sample origin showed KZ and US populations to have similar levels of expected heterozygosity and the average number of alleles per locus. The results of breeds pooled within country suggest that there was no difference between countries for these diversity measures using this set of neutral markers. This finding suggests that populations' geographically isolated from centers of domestication can be more diverse than previously thought, and as a result, conservation strategies can be adjusted accordingly. Furthermore, these results suggest there may be limited need for countries to alter the protocols for trade and exchange of animal genetic resources that are in place today, since no one population has a unique set of private alleles.

This work evaluated different methods for sheep embryo cryopreservation by vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. Both treatments used a vitrification solution (VS) containing 20% ethylene glycol, 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in Dulbecco's phosphate buffered saline (DPBS) with 10% BSA. The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing 25 super-stimulated ewes, 109 transferable morulae were harvested; 35 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 36) or SCURV (n = 38), respectively, thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with expositions 2 and 3 min accordingly. After thawing embryos, only good-quality embryos were transferred. Statistical analyses were performed with Student's t-test. The lambing rate following transfer of fresh, frozen-thawed vitrification and SCURV methods were 18, 12, 14 lambs accordingly. No statistical difference was found for the percentage of does lambing following transfer thawed after vitrification (33.4 ± 5.2a%) and SCURV methods (36.8 ± 6.3b%). The survival rate following transfer of fresh embryos (51.4 ± 4.8c) was higher and in line with previous findings using VS. Differences were statistically significant (ac,bc P < 0.05). Importantly, our data suggest that the HSV Kit can be used to produce viable morulae for implantation as the SCURV, and to as vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep embryos.

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, have stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal’s body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). Ovaries from 19 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% fetal bovine serum, 5 μg mL−1 FSH, 5 μg mL−1 LH, 1 μg mL−1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then, oocytes were placed for 10 min in medium with Hoechst 33342 (3 μg mL−1) and cytochalasin B (7 μg mL−1) to facilitate enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into a cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation, the oocytes were activated for extrusion of the second polar body in 1 mg mL−1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL−1). After activation, the MPII were washed and cultured for 20 h. The control group received the same treatment but were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed that expulsion of the second polar body after activation was observed in more than 59.7% of the MPII that were not vitrified (control group), whereas 37.7% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

Computer-assisted sperm analysers have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Ram semen was collected using electro-ejaculation from 10 adult rams of Chingizskaya indigenous sheep breed. Motility was determined using computer-automated semen analysis (Hamilton Thorne Motility Analyzer, Beverly, MA, USA). Trehalose solution (0.375 M) was added to Tris-buffered saline solution to give the following trehalose extenders: 25, 50, 75, and 100% (vol:vol), and analysed for motility using computer-automated semen analysis. The sperm pellets were resuspended at 24°C in cooling extender – trehalose extenders of each concentration containing 5% egg yolk. The diluted semen was cooled to 5°C within 2 h. The semen was then further diluted 1 : 1 with freezing extender – each trehalose extender containing 1.5% glycerol to obtain a sperm concentration of 2.0 × 108 cells mL–1 – and then loaded into 0.5-mL straws. Straws were frozen using a programmable freezer with a freezing curve of 5°C to –5°C at 4°C per min, –5°C to –110°C at 25°C per min, and –110°C to –140°C at 35°C per min, and then the straws were plunged into liquid nitrogen for storage. Frozen samples were thawed in a 37°C water bath for 30 s and analysed for motility using computer-automated semen analysis. Statistical analyses were performed with a Student's test. The fresh semen samples showed the next results: motility 88.3 ± 2.4%, progressive motility 26.8 ± 6.9%, and progressive velocity 61.9 ± 4.2 μm s–1. Motility of the frozen-thawed spermatozoa was 63.6 ± 2.9% (25% trehalose), 55.6 ± 5.2% (50%), 32.4 ± 4.7% (75%), and 23.6 ± 3.2 (100%). Progressive motility was 15.6 ± 3.9% (25%), 13.7 ± 3.7% (50%), 4.5 ± 1.3% (75%), and 5.2 ± 1.3% (100%). Progressive velocity was 93.5 ± 8.3 μm s–1 (25%), 85.4 ± 8.1 μm s–1 (50%), 65.7 ± 6.1 μm s–1 (75%), 35.2 ± 3.3 μm s–1 (100%). Motility of the frozen-thawed spermatozoa significantly decreased with increasing concentrations of trehalose in the extender (P < 0.05). These preliminary studies showed that further research is needed of use trehalose for ram spermatozoa cryoconservation.

This work evaluated different methods: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The goat morulae were cryopreserved into the High Security Vitrification (HSV) Kit (Cryo Bio System, Paris, France). The vitrification method was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution [VS; 20% (3.6 mol L–1) ethylene glycol (EG), 20% (2.4 mol L–1) dimethyl sulfoxide (Me2SO), and 0.5 mol L–1 of sucrose in Dulbecco's PBS (DPBS) with 10% BSA in both methods]. In our experiment, we used the Vit-Master™ apparatus (MTG GmbH, Bruckberg, Germany). The supercooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing of 30 superstimulated (1200 IU of Folligon, Intervet International, Boxmeer, the Netherlands) goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by HSV Kit and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 M, with exposures of 2 and 3 min, accordingly. After thawing embryos, only good-quality embryos were transferred. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 25, 17, and 19 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer of thawed embryos after vitrification (36.2 ± 4.4%a) and SCURV methods (38.7 ± 6.5%b). The survival rate following transfer of fresh embryos (60.9 ± 5.3c) was higher and in line with previous findings using VS. Differences were statistically significant (ac, bc: P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopreservation of goat morulae as the vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV, a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving goat embryos.