Artificial insemination in cattle

Artificial insemination in the pig

A phantom is used for support of the boar during ejaculation, and the semen collection is performed using the gloved-hand technique, which involves a steady firm pressure applied to the spiral-shaped portion of the penis. The ejaculate of the boar is fractionated, and so collection is normally done through a filter cloth to avoid mixing the more gelatinous portion with the sperm-rich fraction (Fig. 21-1).

Fig. 21-1: Collection of semen from a boar through a filter cloth.

Artificial insemination has become gradually more widespread in countries with intensive pig production. The major drawback has been the relatively poor survival of boar spermatozoa after cryopreservation; they survive much more poorly after freezing than after storage at 16–18°C. Thus, diluted fresh semen is generally used and this poses logistical problems for both its production and distribution. The poor survival of cryopreservation by boar spermatozoa is primarily due to their high susceptibility to cold shock, which may be related to the lipid composition of their membranes and their relatively high content of cholesterol. Insemination doses from the boar are usually packaged in tubes or plastic bags containing about 2–3 × 10⁹ spermatozoa in 70–80 ml.

Insemination of the sow or gilt is easy because the porcine cervix presents neither a portio vaginalis nor circular plicae. Instead, the vagina gradually leads into the cervix through a funnel and, in the cervical canal, the pulvini cervicales create a softer closure. This allows the insemination catheter to be positioned with its tip firmly interlocked with the pulvini cervicales, just as the spiral-shaped portion of the penis would be during copulation. Interestingly, it is now clear that stimulation of the sow or gilt by massage and riding on the back improves the results of insemination by helping the uterus transport semen to the oviducts. Often, a repeat insemination is performed if oestrus is sufficiently prolonged (see Chapter 2).

Artificial insemination in the horse

In the horse, the stallion is allowed to mount a phantom or teaser mare and semen is collected using an artificial vagina. The gel fraction and debris of the ejaculate should be removed using a nontoxic filter. The collected semen can be used fresh, cooled (5°C), or frozen-thawed, using appropriate extenders. Typically, an insemination dose contains 250–500 × 10⁶ progressively motile spermatozoa in a volume of 10–25 ml. For frozen semen, straws of 0.5–4.0 ml are used and insemination doses should contain a minimum of 250 × 10⁶ progressive motile sperm post thawing. The lower number of spermatozoa used for frozen semen allows for production of more straws per ejaculate. Successful use, however, requires that insemination is performed close to the time of ovulation. The semen is normally deposited by catheter in the corpus uteri, using a lubricated gloved finger in guiding the tip of the insemination catheter into the cervical lumen. For specialized purposes (e.g. when using sex-sorted semen) low-dose insemination close to the ostium tubae uterinae can be accomplished through a hysteroscope. The wide opening and lumen of the cervix in the mare provides easy passage of an insemination catheter or hysteroscope into the uterus.

Artificial insemination in small ruminants

In the ram and buck, semen is collected through an artificial vagina as from the bull and stallion. A female, preferably in oestrus, or a phantom can be used for the male to mount. Ejaculation is completed within seconds and the ejaculate is low in volume, but high in concentration of sperm, in both species. Semen can also be collected by electro-ejaculation. The collected semen can be used either fresh or frozen–thawed using appropriate extenders. Typically, an insemination dose contains 300–500 × 10⁶ progressive motile spermatozoa for intravaginal insemination, 100–200 × 10⁶ progressively motile spermatozoa for intracervical insemination, or 50 × 10⁶ sperm for intrauterine insemination. In the ewe the semen is deposited either in the cranial part of the vagina or in the cervical canal. The method of direct intrauterine insemination using laparoscopy has been developed to overcome some of the difficulties of intravaginal and intracervical insemination, especially by reducing the number of spermatozoa required. In the goat the semen may be deposited either intracervically or into the uterus, with less difficulty than in the ewe.

Artificial insemination in the dog and cat

In the dog, semen is collected into a wide-mouth glass or plastic vial or a disposable semen collection cone by digital manipulation. The penis is massaged through the prepuce until an erection of the bulbus glandis and pars longa glandis occurs. The prepuce is then moved caudally so that both the bulbus and pars longa glandis of the penis are exposed. Tightening the grip, the penis is then rotated 180° caudally, keeping the dorsum of the penis dorsal. Only the second fraction of the ejaculate is collected; the first and third fractions consist of prostatic fluid and are discarded. The semen is collected in the presence of a teaser bitch, preferably in oestrus. At insemination, the semen can be placed into either the vagina or the uterus. Intravaginal insemination is uncomplicated and is most commonly used. However, with cryopreserved semen intrauterine insemination is preferred. This can be done either transcervically using a vaginoscope, or surgically through the uterine wall by laparoscopy or laparotomy. Surgery may become necessary due to problems caused by the dorsal position of the portio vaginalis and difficulties in immobilizing the cervix.

Collection of semen from a tomcat requires extensive training and patience, and the presence of a receptive female. Semen can be collected in a tiny artificial vagina customized for rabbits. Alternatively, semen can be collected using electro-ejaculation after sedation. Insemination is intravaginal.

Sex sorting of spermatozoa

Livestock owners have for years sought methods for predetermining and controlling the sex of offspring in their herds for economic reasons. The obvious example is in the dairy industry, where heifer calves are far more valuable than bull calves.

The ability to separate and sort functional spermatozoa according to whether they carry the X- or the Y-chromosome became a reality in the 1990s, based on their different DNA contents (Fig. 21-2). When stained with the DNA-binding dye Hoechst 33342, X-chromosome-bearing spermatozoa absorb more of the dye than do Y-chromosome-bearing. As a consequence, the X-chromosome-bearing spermatozoa will emit more light than will the Y-chromosome-bearing when excited by a laser in a flow cytometer. In the flow cytometer, this difference is measured extremely rapidly in individual stained spermatozoa, each in its separate micro-droplet formed by pumping the semen sample through a special nozzle at high pressure. An electromagnetic charge (positive, negative, or no charge) is then applied to each micro-droplet depending on the DNA content of the spermatozoon within the droplet, and these can then be directed to one side or the other depending on charge. Furthermore, selection of only viable, membrane-intact spermatozoa is made possible by staining with a food dye. Two defined populations of spermatozoa are therefore achieved after flow cytometric sorting using this method; one vial primarily containing viable X-chromosome-bearing spermatozoa, and another primarily containing viable Y-chromosome-bearing spermatozoa. The method has provided a means of determining the efficacy of any sperm sex-sorting enrichment approach, and is the basis for the sperm sexing system that is now used commercially to predetermine the sex of offspring in several species, primarily cattle. Flow cytometric sex-sorting of spermatozoa according to their DNA content is patented and is sub-licensed for mammals (non-human) to XY Inc., through Colorado State University. Species variation in factors such as DNA content and sperm head size make it necessary to optimize sorting conditions species by species to achieve acceptable results. In cattle, the sperm sexing efficiency is about 90% in a commercial setting.

Fig. 21-2: Sorting of X- and Y-bearing spermatozoa by flow cytometry.

Multiple ovulation and embryo transfer in cattle

Embryo recovery

Embryos are recovered 6–8 days after the onset of oestrus (Fig. 21-3). At this stage most embryos will have passed through the oviduct and entered the uterine horns, still surrounded by the zona pellucida. They are therefore accessible for collection through a non-surgical flushing of the uterine cavity. At this stage the embryos are fairly tolerant to handling, not only during their recovery but also in additional procedures such as cryopreservation (see later) or biopsy to isolate cells for sex determination (Fig. 21-4).

Fig. 21-3: Embryo transfer in cattle. Embryos at the morula to blastocyst stage are collected from a donor cow and transferred to synchronized recipients after cryopreservation by either slow-rate freezing or vitrification.

Fig. 21-4: Isolation of a sample of trophectoderm by biopsy of a bovine blastocyst for PCR detection of its Y-chromosome content. The embryo may be transferred directly or cryopreserved while the analysis is performed.

The procedure for embryo recovery. Embryos are collected by flushing, either both uterine horns at one time or each horn separately, using a special transcervical catheter with an inflatable cuff to occlude the lumen of the horn. The most commonly used flushing medium is modified phosphate-buffered saline (PBS) that contains glucose and pyruvate, substrates that are beneficial for post-compaction bovine embryonic development. The phosphate buffer helps keep the pH of the medium stable under practical conditions. Other additions are usually serum or bovine serum albumin (BSA) to provide nutrients and to prevent the embryos from sticking to plastic surfaces during the flushing and handling, and some form of antibiotic to minimize the risk of uterine infection. Epidural analgesia is routinely used during the non-surgical collection; it facilitates rectal manipulations of the genital tract and the collection catheter, and makes the animal stand more quietly both by reducing her discomfort and because she senses reduced control of her hind legs. Flushing fluid is introduced into the uterine cavity until, by rectal palpation, the uterus is judged to be sufficiently distended, at which point the fluid is drained into a collection vessel. The filling and emptying is repeated several times with additional fluid to ensure that the cavity is properly flushed. The combined flushes are filtered through a mesh that retains the embryos but allows debris and blood cells to pass. After this filtration, the reduced volume of flushing fluid above the mesh (and appropriate rinses) must be searched for embryos, using a stereomicroscope.

The results of embryo recovery. The rates of embryo recovery after superovulation vary widely, influenced by factors already described. However, the overall results can be summarized as follows:

• Approximately 5% of the treated donors are not flushed due to a poor response to the superovulatory treatment.

• No transferable embryos are recovered from 20–30% of the flushed donors either because no ova/embryos were recovered, or because none of those recovered were transferable.

• The average total number of ova/embryos per flushed donor is 8–10.

• The average total number of transferable embryos per flushed donor is 5–6.

Multiple ovulation and embryo transfer in pigs

Hormonal stimulation to increase the number of ovulations is not often used because the inherently high ovulation rate in pigs makes it unnecessary. Hormonal interventions are, however, used for synchronization purposes in timing embryo collection. This is especially true when young pigs (gilts) are used; in sows, the natural synchronization following weaning is often adequate.

Embryos are recovered surgically, under full anaesthesia and using a flank or midline approach to the uterus. The uterine horns are flushed through a flared tube or cuffed catheter inserted through an incision or bluntly made puncture, and the embryos are collected and handled as described for cattle.

Pig embryos are generally considered to be much more sensitive to handling than are cattle embryos. This limits the number of treatments that they will tolerate. However, the last decade has brought about significant improvements in this situation, especially in cryopreservation (see later).

The transfer of embryos is also performed surgically under full anaesthesia but instruments for non-surgical transfer are under development.

The naturally high reproductive capacity of the pig has dampened the interest in developing embryo transfer technology in pigs compared to cattle. Still, the technology is of considerable usefulness, both industrially and for research, for reasons similar to those explained for cattle. These include allowing movement of genetic resources with enhanced animal welfare, minimal risk of disease transmission and reduced transportation costs in comparison with transport of live animals. However, it is only during the last decade that application of the technology has started to expand, thanks to several technical advances, and also stimulated by the increasing use of pigs in medical research as a model animal for certain human diseases.

Multiple ovulation and embryo transfer in horses

Horses are very difficult to superovulate, for both physiological and anatomical reasons; the anatomy of the ovaries may hinder ovulation of larger numbers of follicles than usual over a short time period. Therefore, most embryo recoveries are from normally cycling mares.

Embryo collection, handling and transfer are basically as described for the cow because of the similarities in animal size and anatomy of the reproductive organs.

The rejection of all modern breeding techniques in some of the most prominent horse-breeding societies has limited the acceptance and practice of equine embryo transfer. However, that situation is changing and, in recent years, the numbers of transferred embryos have increased, though they remain much lower than in cattle. It is expected that this expansion will continue, fuelled by various breakthroughs in other reproductive technologies.

Multiple ovulation and embryo transfer in small ruminants

Superovulation is performed according to the same principles as for the cow, with synchronization often accomplished with intravaginal sponges or plastic devices that release progesterone.

Embryo collection and transfer are currently done surgically because of the small size of the animals. However, attempts are being made to develop non-surgical embryo transfer methods.

In sheep and goats, multiple ovulation and embryo transfer has been an established technology for many years. It is an essential component of the international trade of genetic resources, facilitates the conservation of endangered species or breeds, and provides an additional genetic tool for dairy breeders owning highly productive flocks that are near the limit of genetic gain attainable through the use of artificial insemination.

Multiple ovulation and embryo transfer in carnivores

Though not used very much in these species, embryo transfer has been applied successfully under special circumstances of commercial interest, for experimental purposes and for preserving endangered species (tigers, for example). In spite of these successes, all aspects of the procedures still require much improvement to make them more practicable.

In vitro production of embryos in cattle