Semen Characteristics and Artificial Insemination in Yak

Summary

The major achievements in scientific research (since 1980) concerning artificial insemination (AI) in yaks are summarised. Semen characteristics and morphological characteristics of yak spermatozoa are described. Semen collection in yaks, including training bulls for restraint and ejaculation into an artificial vagina, are included. Estrus detection, when, where and how to inseminate are all described. Protocols for freezing and thawing semen are also included.

Semen Characteristics

Ejaculates of adult yak bulls are milky-white in colour, pH 6.4 - 6.7 and a volume of 2 - 5 ml. Spermatozoa are present at a concentration of 7.5 - 16.0 × 108/ml, with 70 - 85% progressively motile, 5.1 - 10.0% morphologically abnormal, and 71.8% with intact acrosomes. The general survival time of spermatozoa and the survival index are 54 hours and 18.72 hours, respectively, at 15 degrees Celsius. The resistance index of sperm is 5899 and the viscosity of semen is 1.17 centipoise, lower than semen from Bos taurus bulls (1.92 centipoise). The total nitrogen content of yak semen is 1334 mg/100ml, higher than for Bos taurus semen (877 mg/100 ml) [1-3].

The morphology of yak sperm is similar to that of other cattle [4,5]. The reported dimensions are: sperm head 7.74 - 8.1 μm long and 4.1 μm thick at its widest point ; midpiece 14.47 μm long; and principal piece 49.67 μm.

Artificial Insemination

Semen Collection

Methods of semen collection with an artificial vagina have been established in yaks [1,4,7-9] (Fig. 1). Yak bulls can readily be trained to provide semen for AI. However, due to a lack of information regarding the genetic merit of young yak bulls, most of the semen is collected from older, proven bulls that are considered superior.

Figure 1. Semen collection from a yak bull by artificial vagina.

The training of adult bulls is an important aspect of AI practice. Under natural conditions, bulls remain alone or in groups of 3 - 5 in the high mountains for extended intervals (in the non-breeding season) and they join the female herds only during the breeding season. The first step in training is trying to eliminate the bull’s hostility. Initially, the bull is confined in a pen until it is accustomed to being restrained, fed in a fixed placed and having the herdsman approach. To obtain acceptance by the bull, the herdsman will initially tempt the bull in the pen with grass. Later he strokes and scratches the bull from the front to the rear of the body, and from the back to the abdomen. Since Yaks have a great aversion to being touched on the head, the herdsman avoids this area. When the bull is more accustomed to being handled, the herdsman will start to stroke the scrotum and testes of the bull, to pull on its sheath, and to lead a haltered bull to its feed [9].

Training for semen collection follows by introducing the bull to a female in estrus (whilst she is restrained in a crate) and allowing him to mate. Thereafter, the sheath is held to guide the penis into an artificial vagina (internal temperature, 39 - 42 degrees Celsius). Eventually, a dummy cow can be used for the bulls to mount. Yak bulls should be handled in a gentle manner and their surroundings should be quiet and familiar [9].

Semen can be collected twice weekly. The volume and the quality of the ejaculate are affected by bull age and season [16]. The average volume obtained by artificial vagina is 4.8 ml, with volumes ranging from 2.6, 4.0, 4.4, 5.5, 7.6, and 6.0 ml collected from bulls that were 2 - 8 years old [2]. The highest ejaculate volume (6.3 ml) was obtained in October [2].

Semen Evaluation

Semen evaluation should be rapid but careful, so that samples can be processed to preserve quality and fertility. Inferior samples should be discarded. Although no single test is an accurate predictor of the fertility of individual ejaculates, when several tests are combined, ejaculates with a higher fertility potential can be selected. The major criteria considered include ejaculate volume, sperm motility, sperm concentration, proportion of abnormal sperm, proportion of sperm with an intact acrosome, general survival time, and survival index of sperm.

Insemination

Estrus Detection

Ovulation occurs approximately 12 hours after the end of estrus. Estrus behavior is more commonly displayed at cooler temperatures (i.e. in the morning and evening), especially if the weather includes rain and wind. Signs of estrus are subtle and therefore close observation, especially in the morning and evening, is important. If the females stand to be mounted by teaser bulls, they should be inseminated [10-13].

Insemination

The rectovaginal method is used for insemination (Fig. 2). In the yak female, the cervix is near the vaginal orifice (22.5+/- 0.6 cm), and the uterus has limited mobility. Therefore, the rectovaginal technique is even simpler and easier than in other cattle [10].

Figure 2. Insemination of female yaks.

Dosage

Regardless of the type of semen that is used (undiluted or diluted, pellet or straw), it is generally recommended that females are inseminated twice, 12 hours apart, with 10 million progressively motile spermatozoa used (for each insemination).

Optimum time for insemination

Ovulation occurs about 12 hours after the end of estrus in yaks and the optimum time to inseminate is approximately 10 hours before ovulation. The first insemination is generally performed 24 hours after the beginning of estrus and repeated 12 hours later. Xie [14] reported a conception rate of 82.3% (296/372) following two inseminations, the first at the end of estrus and the second 12 hours later. However, in that report, when the females were inseminated only once (8 hours after the end of estrus), the conception rate was 81.8% (203/248), indicating that a single, appropriately timed insemination can result in fertility comparable to two inseminations.

Insemination site

Xie [15], using the rectovaginal method of insemination, semen was deposited at different sites and fertility was compared. In that study, conception rates were 44.0% (113/302), 52.0% (258/496), 69.6% (162/230), and 50.8% (93/118) when semen was deposited intracervical (1 - 3 cm beyond the external os), intracervical (3 - 5 cm beyond the external os), in the uterine body or in the uterine horn, respectively. Therefore, deposition of semen into the uterine body resulted in the best fertility. It is noteworthy that the cervix is approximately 5 cm long, has rings (similar to other cattle) and the uterine body is rather short (approximately 2 cm).

Frozen Semen

Advances in sperm cryopreservation have played an integral role in cryobiology. Mechanisms of cryoprotection or cryodamage at the molecular and macromolecular levels have been studied with spermatozoa, with ongoing studies to make further improvements. Du [7], Li [8], Zhang [1], and Guo [21] reported methods for freezing semen (in pellets) collected from domestic and wild yaks, respectively.

Extenders

Extenders should be prepared aseptically and used within a week (if fresh) or frozen. Both egg yolk and yak milk are used to protect against cold shock and glycerol is added as a cryoprotectant. Penicillin and streptomycin (or other combinations of antibiotics) are added to inhibit bacterial growth. Extenders used are listed in Table 1.

Diluting, Cooling and Balancing

Fresh semen can be diluted by either of the following two methods. Regardless of the method used, make sure that semen and extender are properly mixed.

One-step Dilution

The semen and the extender should be at the same temperature (32 - 35 degrees Celsius). The ratio of semen to extender depends on sperm concentration and motility but generally ranges from 1:2 - 1:8 (insure a minimum of 10 million progressively motile spermatozoa per pellet). Put the diluted semen in a container, wrap the container in 8 - 16 layers of gauze, and place into a refrigerator (temperature, 4 - 5 degrees Celsius) for 3 - 4 hours [1,3,7].

Two-step Dilution

Fresh semen is initially diluted with Extender I (does not contain glycerol) at a temperature of 32 - 35 degrees Celsius and left at room temperature (approximately 15 - 20 degrees Celsius) for 1 - 2 hours. Then, Extender II (containing glycerol) at room temperature (or at 0 - 4 degrees Celsius) is added and the mixture put into a cold environment (0 - 4 degrees Celsius) for 3 - 4 hours [1].

Freezing

Chilled semen (prior to freezing) should have a minimum of 60% progressively motile spermatozoa. During the preparation of pellets, keep the extended semen cool (place the flask on ice blocks if necessary). For initial freezing, a fluon plate, copper gauze or aluminium plate is placed 1 - 2 cm above liquid nitrogen, the surface free of frost, and the temperature between -80 and -130 degrees Celsius. Each 1 ml of extended semen should yield 10+/- 1 pellets. Complete 100 pellets within 3 min; plunge the pellets in the liquid nitrogen when the last pellet turns from yellow to white. On completion, calculate the number of prepared pellets, check, pack and register.

Thawing of Frozen Semen

The following three regimens are frequently used. The first is 2.9% sodium citrate solution, the second is glucose-sodium citrate solution (5 g of glucose and 0.5 g of sodium citrate dissolved in 100 ml of twice-distilled water) and the third is fresh defatted milk solution. Fresh milk is boiled and cooled, the cream is skimmed off, and the milk filtered through four layers of gauze [17,18].

The inseminator prepares the aliquots by adding thawing solution into sterilised test tubes (an insemination dosage is 1 pellet of semen and 1.5 - 2.0 ml of thawing solution). Then, the tubes are put into a beaker (or a cup) filled with boiling water. The temperatures of the thawing solution should be monitored with a thermometer; when it reaches 38 - 42 degrees Celsius, a semen pellet (held with a bamboo clip or metal forceps) is placed into the tube, the tube is gently agitated, and 3 - 5 seconds later the tube is placed into a water-bath containing water at 30 degrees Celsius. Sperm motility should be rapidly examined and if it exceeds 30%, the sperm should be inseminated as soon as possible.

Packaging, Labelling and Storage

One hundred frozen sperm pellets are packed in a sterilised bottle made of polythene, the bottle must be labelled (bull identification, freezing date, quantity, lot number, producer, etc), and stored in liquid nitrogen [20]. The application of AI, especially with frozen semen, can dramatically hasten genetic progress and have substantial economic benefits. For example, experiments conducted on Waqie farm (Sichuan Province) showed that high milk production is achieved in F1 hybrids (Yak females and Holstein semen). The meat yield of crossbreeds also increased substantially. Birth weights of F1 males and females were 73.7 and 76.3%, respectively, greater than that of purebred yaks, and the body weight at 18 months of age (229 kg) surpassed the average weight of mature yaks (5 years, 222 kg). Zhang [9] reported that until September 1997, 40 stud yak bulls were producing 400,000 semen pellets frozen which amounted to about 2 000 000 RMB Yuan annually. Frozen semen could be used to inseminate 200,000 cows with a conception rate of 80% (35% higher than that achieved with natural service), resulting in an additional 70,000 calves and 1.1 x 106 Kg butter produced compared to traditional mating and production systems. These improvements would result in a total increase of 27 million RMB yuans annually.

Although AI has not been widely adopted on a widespread basis in the vast area in which Yaks are raised, there is considerable optimism that AI, and in particular the use of frozen semen, will be more widely used in the future.