Reproductive Biotechnologies: Current Status in Yak Reproduction

Summary

Novel reproductive characteristics of yak and expansion of commercial yak production have generated considerable interest in the application of reproductive biotechnologies in yak, both for research and increased commercial production. The objectives of this presentation are to review the current status of estrus synchronization, superovulation, and in vitro embryo production.

Introduction

For several years, there has been considerable interest in the application of reproductive technologies in yak, both for research and improved productivity. As commercial yak production has expanded, reproductive technologies that are used in domestic cattle (e.g., pregnancy diagnosis, artificial insemination, estrus synchronization, embryo transfer, and the collection and preservation of semen) have been attempted in yak. Although similarities between Bos taurus cattle and yak have facilitated the direct application of these technologies to the latter, critical differences in biology and husbandry have limited application and success.

Reproductive studies in yak have shown that anatomically and physiologically they are generally similar to cattle. One notable difference is that yak are apparently seasonally polyestrus. Depending on the report, yak cycle from late summer to late fall or from July to September; the short breeding season is dictated by intense breeding activity during the early part of the breeding season and subsequently the absence of conception due to declines in both temperature and feed supplies. In addition, yaks have a relatively small uterus and ovaries (comparable in size to young beef heifers). Consequently, ovarian follicles and corpora lutea are smaller and more difficult to detect by rectal palpation than they are in cattle [1,2]. Furthermore, delayed maturity, silent estrus, low conception rates, and prolonged postpartum anestrus in yaks seriously limit both reproductive performance and the application of reproductive technologies that are widely used in cattle.

Estrus Synchronization

Estrus synchronization in the yak has been effectively accomplished with products commonly used in cattle, including PGF2α and progesterone. Synchronization of estrus is most effective during the breeding season but estrus can be induced outside the breeding season. Regardless of the method used, considerable effort must be made to minimize stress (e.g., due to handling) or fertility will be adversely affected.

When PGF2α is used, the animal must be cycling and have a responsive corpus luteum. Following a single injection of PGF2α (during the ovulatory season), approximately 50 % of yaks will be detected in estrus (similar to cattle), but the interval from treatment to estrus is usually slightly longer than in cattle. A second injection of PGF2α 11 days after the first will effectively synchronize the group. In one study [3], 135 yaks were injected twice-daily with two PGF2α preparations. Most yaks (55 - 70 %) were detected in estrus 3 days after treatment. However, there was a seasonal effect on the proportion in estrus, with 50.0 % and 46.2 % detected in estrus following administration of Oestrophan and Enzaprost, respectively, in July and a better response rate (61.5 % and 58.3 %) in August. Following two treatments at 10-day intervals, the estrus synchronization rate 86.2 % and 90.0 % and the conception rate following insemination with frozen-thawed Holstein semen was 77.9 and 78.9 % (compared to 85.7 % in a control group). Overall, these results were similar with those reported in cattle [3,4].

A steroid preparation ("Three-in-One" containing testosterone propionate, progesterone, and estradiol benzoate in concentrations of 25, 12.5 and 1.5 mg/ml, respectively) has been used to synchronize estrus in yaks. In one study [6], 66/93 (71.0 %) of yaks treated with this preparation were detected in estrus within 5 days, compared to 13/90 (14.4 %) in control group, with conception rates of 22/71 (31.0 %) and 21/78 (27.0 %), respectively. In a subsequent study [7], the same preparation was given at the following doses: 1.25 ml and 0.75 ml/100 kg body weight. In this experiment, 18, 12 and 6 yak cows, respectively, were detected in estrus 96 - 120 hours after treatment and a total of 36, 24 and 12 (48/150 = 48.0 %) were detected in estrus 24 - 120 hours after treatment. In another study [5], 11/17 (64.7 %) of females were detected in estrus following treatment with this preparation, compared to 15/26 (57.7 %) that were detected in estrus within 2 - 4 days after treatment with PGF2α.

Superovulation

Superovulation has been attempted in yak females using a variety of treatments and under varying conditions. Satisfactory responses have not been consistently achieved, thereby limiting the use of embryo transfer in this species. In a small trial [8], 3 barren females (4 - 8 years old) were given a CIDR (containing 1.2 g progesterone) for 12 days, injected with Lutalyse(PGF2α) on the third day after the first injection of FSH and inseminated 24 - 36 hours after the last injection of FSH. The average number of luteal glands and follicles were 5.0 +/-  0.6 and 1.3 +/-  0.9, respectively, but embryo recovery was not reported.

Davaa et al. [9] used FSH and PMSG to induce superovulation in yak cows. Estrus was detected 34.1 +/-  0.52 hours after the prostaglandin treatment. The average number of ovarian follicles was 5.4 +/-  0.65 and 4.5 +/-  0.43 of them ovulated (2.6 +/-  0.30 and 1.9 +/- 0.20 ovulated from the right and left ovaries, respectively).

In Vitro Embryo Production (IVP)

Difficulties and inconsistencies associated with in vivo embryo production in yaks may be overcome with in vitro maturation and fertilization techniques. Potential advantages include:

1. Circumvention of the need to synchronize ovulation for AI;
2. The potential for producing more embryos that can usually be collected from hormonally stimulated donors;
3. The ability to make use of animals with certain types of infertility (e.g., endometritis or oviductal occlusions);
4. A reduction in the numbers of viable sperm needed;
5. Using sperm microinjection techniques, the potential for using nonmotile, non viable sperm, or epididymal-derived sperm for assisted fertilization;
6. The potential for salvaging genetic material from female animals after death; and
7. The possible utilization of pre-pubertal or pregnant animals as oocyte donors.

Despite success in other species, there are no reports of yak calves being produced from following in vitro maturation and fertilization of oocytes collected from nonstimulated females. However, it is noteworthy that these embryos are capable of developing to the blastula phase in vitro.

Using standard procedures, Chen et al. [11] studied the efficiency of IVP methods in yak and local breeds [10]. Ovaries (8 and 50 from yak and local cattle, respectively) were collected after slaughter, washed with saline or PBS, maintained at approximately 30°C, and transported to the laboratory within 3 hours. Ovaries were rinsed with 75 % alcohol, washed with saline and the follicles were aspirated with a syringe. The aspiration media was TCM-199 with 25 mM HEPES and 2 % calf serum. It was noteworthy that an average of 7.0 and 2.7 oocytes were obtained per ovary from yak and local breed cows, respectively. Only oocytes with complete granular cells were selected for maturation culture; they were washed three times with maturation media and cultured for 24 hours. The addition of serum (15 %), FSH, LH, and estradiol to the maturation media enhanced the maturation rate.

Sperm quality and the induction of sperm capacitation in vitro also limit IVP in yak. Only recently have morulae and blastocysts been produced following in vitro maturation (IVM) and in vitro fertilization (IVF) of yak oocytes. In certain circumstances, epididymal sperm for IVF may obviate the need for supplemental treatments (e.g., calcium ionophore) to induce capacitation in vitro. Cryoprotectants appropriate for freezing yak semen may not be effective for epididymal sperm. An alternative to cryopreserving epididymal sperm may be the direct refrigeration of testes, which for certain animals, can maintain sperm viability for up to 5 days [10].

Chen et al. [11] used epididymal sperm from yaks to study sperm capacitation conditions. Frozen (in a pellet) semen from local breed was included as a comparison. Yak testes were collected soon after slaughter; the cauda epididymis was excised, minced, and put into dishes containing PBS. Sperm samples with acceptable motility and density were sealed in a tube and kept in a fridge (4°C). Samples were subsequently removed and mixed with 1 ml of BO media containing 10 mM caffeine and 3.6 IU heparin. Motility was assessed and the semen was subsequently washed twice and centrifuged at 350 G for 5 minutes; the sediment was mixed with the BO medium with 0.6 % BSA to adjust sperm concentration and 100 ul droplet was made, covered with mineral oil, and put into an incubator containing carbon dioxide.

Following culture in maturation media for 24 hours, oocytes were aspirated, put into BO media (with 0.3 % BSA) and washed three times. Thereafter, 10 - 15 oocytes were put in one semen droplet, co-cultured for 6 hours, and washed three times (with culture media) to remove extra sperm. Following this washing, 10 - 15 ova were put in each 100 ul culture droplet, covered with mineral oil, and cultured for a further 48 hours. At that time, cleaved ova were counted (and separated from non-cleaved ova), with 2 - 8 cell embryos cultured up to day 8 (and the number that formed blastocysts was determined). Cleavage and blastocyst rates for yak IVF were 35/56 (62.5 %) and 9/56 (16.1 %), respectively, indicating that these procedures, derived from those used in cattle, produced acceptable results that were at least as good as those obtained with IVP using oocytes and semen from local cattle.

Luo et al. [12] obtained ovaries from 4 yaks after slaughter. It is noteworthy that one of these yaks was 40-days pregnant. Ovaries were washed with 0.9 % saline, kept in a vacuum bottle (at 25 - 30°C) and taken to the laboratory within 5 hours after collection. Oocytes were aspirated, washed 3 times with TCM-199 containing 5 % calf serum, and cultured at 38.5°C, 5 % CO2 and 100 % humidity for 20 hours. Frozen-thawed semen from wild yaks was diluted to suspension with BO media containing 5 mM caffeine. The solution was centrifuged twice for 5 mins at 1800 G and the semen concentrations adjusted to 20 million per ml. Capacitation was induced by incubation in Tyrod’s media (with 20 ug/ml heparin) for 15 minutes. Following 20 hours of culture, oocytes were washed with BO media (containing 0.5 % BSA and 10 ug heparin). Semen droplets (50 ul) were made in Petri dishes, covered with sterilized paraffin oil, and equilibrated in a carbon dioxide incubator; mature oocytes were moved into droplet after 1 - 2 hours. For insemination, capacitated semen suspensions were flushed into semen droplets (final concentration, 4 million cells/ml) and the Petri dishes were immediately put into the incubator. After 5 hours of incubation, fertilized eggs were washed with TCM-199, incubated for 48 hours with TCM-199 (38.5°C, 5 % carbon dioxide), the incubation medium was changed and they were incubated (same conditions) for an additional 72 hours. In this study, 25 oocytes were aspirated from 8 ovaries and after incubation for 20 hours, 20 oocytes developed to meiosis II. Fertilization and cleavage rates were 12/20 (60 %) and 8/20 (40 %), respectively. Although 2 normal morale were transferred into the uterus of a Holstein-Friesian cow, she was subsequently diagnosed nonpregnant.

It has been reported that Y-bearing bovine spermatozoa may be distinguished by fluorescent in situ hybridization (FISH), using a specific DNA probe; this technique provides an in vitro verification for experiments producing sex-oriented or sex-specific semen. It was reported that a bovine Y-specific probe produced a visible signal in yak spermatozoa; specific binding of the probe to the corresponding sex chromosome was subsequently confirmed on a chromosome preparation [13].

Limitations and Associated Problems

Under ideal conditions, the utilization of reproductive technologies in yak can produce results similar to those in cattle. However, yak behavior is very different from that of cattle. In that regard, yaks are often difficult (indeed dangerous) to handle, with considerable potential for harm to the handlers as well as the animals.

Research progress has been made is several technologies but success in others has been limited. Although the study of reproductive events in yak has generally lagged far behind that of domestic livestock, recent interest and investigations have in part corrected this deficit. Although traditional approaches to embryo transfer technologies have resulted in limited success, the demonstration that IVP (utilizing techniques developed for cattle), can be very successful yaks provides great optimism. Notwithstanding, there is still much to be learned before these technologies are commonplace in yak. In that regard, short-term preservation of yak ovaries and/or oocytes and cryopreservation of oocytes and embryos would be of considerable benefit.

Remarkably, many techniques developed and proven in cattle have been adapted to yak with only a few, minor modifications. The success of IVP techniques in yak suggests that there are many opportunities for similar approaches in other animals. In that regard, IVM and IVF utilizing epididymal spermatozoa and IVC with oviduct cell co-culture are likely to be of use in many different species in the near future. It is expected that this will promote assisted reproduction in exotic and endangered species and facilitate opportunities for the international movement of gametes.