The equine frozen semen industry.

Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major impediments to the development of the frozen semen industry include 1. Lower fertility with frozen semen as compared to cooled semen for many stallions. 2. Increased costs associated with management of mares for AI with frozen semen using current insemination protocols. 3. Unfavorable marketing practices for frozen semen. Reports of fertility with cooled transported semen in commercial breeding programs indicate seasonal pregnancy rates ranging from 60 to 90%. We compiled data from three commercial transported cooled semen programs in which semen from 16 stallions was used for insemination of 850 mares throughout North America by local veterinarians. During the 1999 and 2000 breeding seasons, first cycle and seasonal pregnancy rates of 59.4 and 74.7% were obtained. During that same period, first cycle and seasonal pregnancy rates of 51.3 and 75.6% were obtained following insemination of 876 mares with frozen semen from 106 different stallions processed by our laboratory and distributed through our commercial distribution program. First cycle and seasonal pregnancy rates were higher for mares bred outside of North America than for mares bred within North America (53.5 and 81.9 versus 49.4 and 65.6%, respectively). Seasonal pregnancy rates were higher presumably because of the better mare management employed for mares bred with exported semen and the fact that some of the domestic mares were switched to cooled semen insemination after a failed first cycle attempt with frozen semen. These data support the position that comparable seasonal pregnancy rates may be obtained using frozen and liquid cooled semen in a commercial setting.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Comparison of conventional and computer-assisted semen analysis in cockatiels (Nymphicus hollandicus) and evaluation of different insemination dosages for artificial insemination

Many psittacine species are threatened in the wild and also rare in captivity. Therefore, successful conservation and breeding programs are important to save these species. Unfortunately, clutches in conservation programs are frequently infertile. Semen evaluation is beneficial to investigate the causes of infertility and is advisable before artificial insemination (AI). In this study, we analyzed the semen of cockatiels (Nymphicus hollandicus) using two different methods and investigated different insemination dosages for AI. Cockatiels (n = 30) were divided into two groups (group A: nine males; group B: six males). The males in group B were endoscopically sterilized, whereas the males in group A were used as semen donors. In the first part of the study, the semen of males in group A was evaluated by semen analysis. Semen samples were collected by the massage technique and examined using a conventional light microscope and a computer-assisted semen analyzer for comparison. Results demonstrated that the evaluations of motility, progressive motility, and sperm concentration, but not of live/dead ratio, correlated strongly for both methods. However, the results for sperm concentration, progressive motility, and live/dead ratio differed significantly. In the second part of our study, the volume and quantity of spermatozoa of the semen samples were adjusted and used for AI of females of group B. Intravaginal insemination with 250,000 spermatozoa resulted in five of 17 (29%) eggs fertilized; however, intracloacal insemination resulted in only four of 57 (7%) eggs fertilized at 232,000 and 250,000 spermatozoa but none at higher or lower dosages.     

Artificial insemination in South American camelids and wild equids

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the "stallion effect", extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.     

Current topics in artificial insemination of sheep

There have been developments in several aspects of artificial insemination (AI) in recent years, some of which have been directly responsible for proliferation of AI in the sheep-breeding industries of several countries. The most notable advances have probably been associated with the development of intrauterine insemination by laparoscopy. There is potential for refinement of some of the related techniques, particularly in the area of control of ovulation and definition of appropriate times and optimum doses of spermatozoa for insemination. It is unlikely that laparoscopic AI will be developed sufficiently that it will become readily affordable, and therefore widely practised, by commercial producers. Unfortunately, there has been little progress in the past few years in improvement of the methods of cryopreservation of ram semen. There is considerable potential for AI to have a significant impact on the genetic improvement of sheep, though this has yet to be evaluated in practice. However, if the full potential of AI in sheep is to be realized, it will likely only happen when methods of freezing semen are improved sufficiently that cervical or even vaginal insemination can be widely used with frozen-thawed semen, or when practicable methods of deep cervical or intrauterine insemination through the cervix are developed.     

Spermophagy in semen in the red wolf,Canis rufus

The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley-Liss, Inc.     

Artificial insemination in domestic cats (Felis catus)

Artificial insemination (AI) in cats represents an important technique for increasing the contribution of genetically valuable individuals in specific populations, whether they be highly pedigreed purebred cats, medically important laboratory cats or endangered non-domestic cats. Semen is collected using electrical stimulation, with an artificial vagina or from intact or excised cauda epididymis. Sperm samples can be used for AI immediately after collection, after temporary storage above 0 degrees C or after cryopreservation. There have been three and five reports on intravaginal and intrauterine insemination, respectively, and one report on tubal insemination with fresh semen. In studies using fresh semen, it was reported that conception rates of 50% or higher were obtained by intravaginal insemination with 10-50x10(6) spermatozoa, while, in another report, the conception rate was 78% after AI with 80x10(6) spermatozoa. After intrauterine insemination, conception rates following deposition of 6.2x10(6) and 8x10(6) spermatozoa were reported to be 50 and 80%, respectively. With tubal insemination, the conception rate was 43% when 4x10(6) spermatozoa were used, showing that the number of spermatozoa required to obtain a satisfactory conception rate was similar to that of cats inseminated directly into the uterus. When frozen semen was used for intravaginal insemination the conception rate was rather low, but intrauterine insemination with 50x10(6) frozen/thawed spermatozoa resulted in a conception rate of 57%. Furthermore, in one report, conception was obtained by intrauterine insemination of frozen epididymal spermatozoa. Overall, there have been few reports on artificial insemination in cats. The results obtained to date show considerable variation, both within and among laboratories depending upon the type and number of spermatozoa used and the site of sperm deposition. Undoubtedly, future studies will identify the major factors required to consistently obtain reliable conception rates, so that AI can become a practical technique for enhancing the production of desirable genotypes, both for laboratory and conservation purposes.     

In vitro fertilization as a predictor of fertility from cervical insemination of sheep

The objective of this study was to determine if the quality of frozen-thawed ram semen could be effectively evaluated through in vitro fertilization (IVF) procedures prior to insemination as a means of improving pregnancy rate. In experiment 1, frozen semen from four Belclare rams was assessed using IVF and was used for cervical insemination of ewes (n = 181) in 13 pedigree Belclare flocks. There was a significant association between IVF score (proportion of oocytes cleaved at 48 h post insemination) and non-return rate (P < 0.001). For experiment 2, semen from nine Belclare rams was evaluated by IVF and semen from rams with the highest (n = 3) and lowest (n = 2) IVF scores was used for cervical insemination of ewes (n = 111) under experimental conditions. Differences in pregnancy rates between individual rams did not reach significance. Experiment 3 was designed to determine if differences detected between rams at field level could be accurately identified via IVF evaluation and involved frozen semen from eight Norwegian rams of known field fertility (non-return rates ranged from 45.7 to 73.8%). IVF score did not reflect the differences in field fertility. In the final experiment six of the eight Norwegian rams involved in experiment 3 were selected based on IVF score (three highest and three lowest) and their semen was used for cervical insemination (n = 90 ewes). While significant differences in pregnancy rate were found between individual rams (P < 0.02, range: 12.9-65.8%) they were not associated with IVF score. Ewe breed had a significant effect (P < 0.003) on pregnancy rate in both experiments 2 and 4. In conclusion, there was no evidence from this study that the evaluation of semen quality through IVF provided a useful predictor of pregnancy rate under field conditions. It may be that the IVF procedures as used routinely, which are essentially designed to maximize blastocyst yields rather than for detecting differences in fertilizing ability between batches of sperm, need to be modified.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis)

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.     

Integration of future biotechnologies into the equine industry

There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on thousands of spermatozoa in a matter of seconds. Development of procedures for insemination of mares with relatively small numbers of spermatozoa has the potential to change how stallions and their semen are managed. This review discusses the use of insemination of fresh, frozen and sex-sorted spermatozoa in relatively small numbers compared with conventional insemination technologies. The recent acceptance of frozen-thawed semen by many of the major breed registries has stimulated an increase in research on frozen semen. Many of the studies have focused on identifying damage during the freezing and thawing process. Numerous studies also have been conducted to modify freezing extenders so that the sperm are protected during the freezing and thawing process. The production of in vitro-produced embryos is extremely limited in the horse due to the failure of in vitro fertilization. However, intracytoplasmic sperm injection (ICSI) has been used for the production of foals from stallions that have less than typical sperm numbers or from stallions that have died and a limited quantity of frozen semen is available. This technique has been used by several laboratories to produce embryos in vitro. The breeder and veterinarian now have access to techniques that allow assessment of semen quality, improvement of procedures for freezing and thawing and insemination of mares with fewer numbers of spermatozoa. It is likely that the next decade will also produce tremendous advances in semen technologies that can be utilized in the horse industry.     

Effects of different lots of semen from the same bull on in vitro development of bovine oocytes fertilized in vitro

The effectiveness of 8 different lots of semen from the same bull on the in vitro development of bovine oocytes fertilized in vitro was evaluated. Cleavage and development rates to the blastocyst stage were not significantly different among the 8 lots of semen. However, the cleavage and development rates varied no more than +/-11 and +/-6%, respectively, in overall rates. A maximum of 35.2 and 27.9% difference in cleavage and development rates to the blastocyst stage, respectively, was observed using a single straw from the same semen lot for insemination in 4 trials. This variation tended to be higher than that observed for the cleavage and development rates of oocytes after insemination with double straws from a single lot. These results indicate that the developmental capacity of embryos after insemination are affected by factors associated with a different semen lot from the same bull and with different straws from a single lot.     

Immunological responses to semen in the female genital tract

When spermatozoa, seminal plasma and semen extender reach the uterus and interact with local leukocytes and endometrial cells, several immune mechanisms are initiated which have immediate, mid-term and long-term effects on ovulation, sperm cell selection, fertilization and pregnancy success by assuring the acceptance of fetal tissues. This report gives an overview on relevant key immune mechanisms following roughly the time axis after insemination. Detailed knowledge regarding these mechanisms will aid maximizing reproductive efficiency in livestock production. In the future, the many species involved will require a more comparative approach, since evidence is growing that endometrial physiology and the response to varying amounts and compositions of seminal plasma, various semen extenders, and variable numbers of spermatozoa also provoke different immune responses.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Artificial insemination in dromedary camels

Artificial insemination (AI) is an important technique in all domestic species to ensure rapid genetic progress. The use of AI has been reported in camelids although insemination trials are rare. This could be because of the difficulties involved in collecting as well as handling the semen due to the gelatinous nature of the seminal plasma. In addition, as all camelids are induced ovulators, the females need to be induced to ovulate before being inseminated.     

Comparative Examination of Capercaillie (Tetrao urogallus L.) Behaviour Responses and Semen Quality to Two Methods of Semen Collection

Artificial insemination (AI) is very helpful in solving the reproductive and biodiversity problems observed in small, closed avian populations. The successful production of fertilized eggs using AI is dependent on the collection of good quality semen. Two methods of male sexual stimulation and semen collection from captive kept capercaillie (Tetrao urogallus L.), one of the most seriously endangered grouse species in Europe, are compared in this study. Ejaculates were obtained either with the use of a dummy female or by the dorso-abdominal massage method. Differences in the individual responses of the males to the two methods of semen collection as well as in their semen quality were noted. Only sperm concentration (432.4 x 10⁶ mL^-1 with dummy female and 614.5 x 10⁶ mL^-1 for massage method) was significantly affected by capercaillie stimulation method. Sperm motility and morphology were not affected (P≥0.05). Thus, for semen collection from captive kept capercaillie both methods can be used successfully. The dummy female can be an alternative to dorso-abdominal massage method, commonly used for semen collection from domesticated bird species.