The spatio-temporal distribution of aromatase cytochrome in ovary throughout the canine oestrous cycle.

CONTEXT: New animal welfare legislation and ethical guidelines encourage alternative approaches for canine contraception, instead of surgical gonadectomy which is considered invasive and unjustified in healthy dogs. AIMS: Reversible contraception might be achieved by inhibition of aromatase (CYP19), an enzyme catalysing the conversion of androgens to oestrogens. This study provides insights into the spatio-temporal expression and distribution of aromatase in canine ovarian tissue. METHODS: Ovarian tissue was collected from 39 healthy and sexually mature bitches during different stages of the oestrous cycle: pro-oestrus (n =8), oestrus (n =12), dioestrus (n =9) (luteal phase) and anoestrus (n =10). Localisation of cytochrome P450 aromatase was determined by immunohistochemistry. KEY RESULTS: Aromatase activity in the dog is high during pro-oestrus, ovulation and early dioestrus. Comparing types of follicles and corpora lutea, the highest aromatase abundance was found in antral follicles and luteinising follicles, whereas corpora lutea and early antral follicles showed an intermediate presence of the enzyme. Interesting was the high abundance of aromatase in luteinising theca interna cells, prevailing over granulosa cells. CONCLUSIONS AND IMPLICATIONS: Understanding of cells involved in oestradiol production is important for targeted inhibition of oestradiol synthesis, possibly offering an approach for contraception and suppression of oestrus.

Potential applications of assisted reproductive technologies (ART) in reindeer (Rangifer tarandus).

Interest in the use of assisted reproductive technology in reindeer husbandry has gradually increased during the last decades. This article reviews Western and Russian literature on reindeer semen collection, semen cryopreservation and artificial insemination. In addition, literature on the synchronisation of the stage of reindeer oestrous cycle among females, recovery of in vivo embryos, embryo transfer, the production of in vitro-produced embryos and pregnancy diagnosis is reviewed.

Reproduction of male reindeer (Rangifer tarandus).

The present review provides a comprehensive assessment on the basic aspects of the reproduction of the Rangifer male with a special focus on pubertal development, and the seasonal dynamics of gametogenesis, androgenesis, antler cycle and other physiological and behavioural characteristics of the rutting male. A greater understanding of the reproductive physiology of the males is needed to improve existing management strategies of semi-domestic stocks in the changing climate and to develop species-specific assisted reproductive technologies for improving the in situ and ex situ conservation programs to preserve endangered subspecies as well as to facilitate the transfer of genetic resources between fragmented wild or semi-domestic populations.

Administration of aromatase inhibitor MPV-2213ad to blue fox vixens (Vulpes lagopus) as a model for contraception in female dogs.

The interest in non-surgical approaches to contraception and fertility control in female dogs has increased in recent years. In this study the effect of an aromatase inhibitor (finrozole) was evaluated in fur production animals, farmed blue fox vixens, as a model for contraception in bitches. A total of 80 vixens were divided into 4 groups, receiving orally placebo (A) or finrozole 0.5 mg/kg (B), 3.5 mg/kg (C) or 24.5 mg/kg (D) for 21 consecutive days beginning in the pre-ovulatory period of heat. Monitoring of the vixens included clinical signs of heat, measurement of vaginal electrical resistance (VER) as well as oestradiol and progesterone concentrations in plasma. The approximate relation of the start of treatment to ovulation varied from 11 days before to one day after ovulation provided that the LH peak occurred 0.5-2 days before the VER peak and ovulation was then estimated to occur 2 days after the LH peak. Seventy vixens were artificially inseminated within 8 h after a 50 Ω decline in vaginal electrical resistance was detected. Ten vixens were not inseminated. Pregnancy was confirmed by transabdominal ultrasound examination and birth of cubs was recorded. The pregnancy rates in the groups were 89.5% (A), 81.3% (B), 55.6% (C) and 52.9% (D). The average number of live born pups in the four groups was 9.4 (A), 7.0 (B), 5.8 (C), and 3.8 (D), respectively. No deleterious effects (for instance malformations) of finrozole on pups could be verified. The administration of finrozole did not have a significant effect on oestradiol parameters and VER values in vixens. Progesterone values were significantly higher in treatment groups compared with the placebo group. The results indicate that pregnancy could be avoided by finrozole provided that doses of ≥3.5 mg/kg were used and the treatment was initiated at least four days before the day of artificial insemination. This corresponds with two to six days before ovulation provided that the LH peak occurred 0.5-2 days before the VER peak and that ovulation then occurred in average 2 days after the LH peak.

Whole blood transcriptome analysis reveals footprints of cattle adaptation to sub-arctic conditions.

Indigenous cattle breeds in northern Eurasia have adapted to harsh climate conditions. The local breeds are important genetic resources with cultural and historical heritages, and therefore, their preservation and genetic characterization are important. In this study, we profiled the whole-blood transcriptome of two native breeds (Northern Finncattle and Yakutian cattle) and one commercial breed (Holstein) using high-throughput RNA sequencing. More than 15 000 genes were identified, of which two, 89 and 162 genes were significantly upregulated exclusively in Northern Finncattle, Yakutian cattle and Holstein cattle respectively. The functional classification of these significantly differentially expressed genes identified several biological processes and pathways related to signalling mechanisms, cell differentiation and host-pathogen interactions that, in general, point towards immunity and disease resistance mechanisms. The gene expression pattern observed in Northern Finncattle was more similar to that of Yakutian cattle, despite sharing similar living conditions with the Holstein cattle included in our study. In conclusion, our study identified unique biological processes in these breeds that may have helped them to adapt and survive in northern and sub-arctic environments.

Effect of education on ability of AI professionals and herd-owner inseminators to detect cows not in oestrus and its relation with progesterone concentration on day of re-insemination.

The effect of training background of persons performing artificial insemination (AI) (herd-owner inseminators (OWNER), AI technicians (AI-T), and fertility consultants (FC)) on pregnancy rate and their ability to detect cows not in oestrus were studied. A total of 1584 re-AI occasions on 754 dairy farms were included. Milk samples for progesterone (P4) analysis in all cases were collected, as were data on the herd, previous breeding attempts, oestrous signs, uterine tone, slipperiness of cervix, and co-operation of the cow. Further breeding attempts and next calving or culling date were sought from registers. The cases were distributed into three categories based on P4 concentrations; <6 nmol/l (no luteal activity, could be in oestrus), 6-10 nmol/l (some luteal activity), and >10 nmol/l (high luteal activity, not in oestrus). Of cows offered for re-AI 7.7% had P4 concentration >10 nmol/l, with no difference between OWNER farms and farms using AI service. OWNERs chose for AI more cows having intermediate P4 than farms using AI service (9.8% vs. 5.9%, p < 0.05). AI-Ts recommended no AI significantly less often than FCs (1.6% vs. 4.9%, p < 0.01). Both groups were equally right: 71% and 68% of cows that were recommended to have no AI had high P4 concentration. Due to courageous and correct rejection of cows with high P4, FCs inseminated proportionally more cows in low P4 and less cows in intermediate P4 than OWNERs (p < 0.05). Of cows finally inseminated, 36.7% became pregnant, with no difference between OWNER farms and farms using AI service. Fertility consultants had higher pregnancy rates than AI-Ts (39.6% vs. 32.6%, p < 0.05). Toneless uterus and sticky cervix at AI significantly correlated with AI occurring at the wrong time (p < 0.001). Behaviour of the cow at AI did not predict P4 concentration. In conclusion, 7.7% of cows offered for re-AI had high P4 concentration. Training of AI personnel increased their ability to detect and reject these cows.

Embryo production with sex-sorted semen in superovulated dairy heifers and cows.

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15-hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30-45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8-14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.

The effect of ascorbic acid during biopsy and cryopreservation on viability of bovine embryos produced in vivo.

Multiple ovulation embryo transfer (MOET) is used to make more rapid progress in animal breeding schemes. On dairy farms, where female calves are more desired, embryo sex diagnosis is often performed before embryo transfer. Fresh transfers have been favored after biopsy due to cumulative drop in pregnancy rates following cryopreservation. The aim of this study was to explore whether exposure to ascorbic acid (AC) during biopsy and freezing increases the viability of biopsied embryos after cryopreservation. Data on presumptive pregnancy and calving rates of biopsied and cryopreserved/overnight-cultured embryos were gathered. Results showed differences in presumptive pregnancy rates between the groups: 45% for both biopsied-cryopreserved groups (control and AC), 51% for biopsied-overnight-cultured embryos and 80% for intact-fresh embryos. Differences between the groups were also apparent in calving rates: 22% for biopsied-cryopreserved control embryos, 31% for biopsied-cryopreserved AC-embryos, 23% for biopsied-overnight-cultured embryos and 63% for intact-fresh embryos. It is concluded that manipulated embryos are associated with lower presumptive pregnancy and calving rates compared with intact-fresh embryos. The highest calving rates for groups of manipulated embryos were achieved in the AC-group. Therefore, addition of AC can be recommended if biopsy is combined with freezing before transfer.

Reproductive technologies relevant to the genome resource bank in Carnivora.

Carnivora is one of the most species-rich order of Mammalia. Some species, e.g. domestic cats, dogs and ferrets, are among the most popular pets; others, such as minks and farmed foxes, have economic value for the fur breeding industry. Still others, such as tigers, bears and other top predators, have great impact on the health of natural ecosystems. Most if not all Carnivora species have great cultural and aesthetic importance for man. There are enormous differences between mammalian species in reproductive physiology, and it is not surprising that reproductive technologies can be used with high efficiency with some animal groups, e.g. most farm animals and laboratory rodents, but are very laborious when used with Carnivora species, which often possess unique reproductive traits. The efficiency of assisted reproductive technology (ART) applied to semi-domestic, non-domestic and especially to endangered species of Carnivora remains extremely low in most cases, and often the first positive result reported is the only instance when ART has been successful with that species. Although there are approximately 270 species in the Carnivora order, to the best of our knowledge, successful published attempts to apply ART have been reported for only four families: Mustelidae, Felidae, Canidae and Ursidae. The main achievements in ART, embryo technology in particular, for these families of Carnivora, together with challenges and problems, are reviewed in the relevant sections.

Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer.

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n=195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n=33), December 2006 (n=92), or January 2007 (n=70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.

Reproduction in the European mink, Mustela lutreola: oestrous cyclicity and early pregnancy.

Despite efforts undertaken to conserve the endangered European mink, its reproduction is still poorly studied. The aim was to study its reproductive cyclicity, faecal progesterone concentration and ovarian changes during early pregnancy, with the emphasis on the pre-implantation period and implantation. During the 2004 breeding season, oestrous cycle was monitored in 39 females as well as ovarian changes during early pregnancy in 22 females. During the 2007 breeding season, faecal progesterone concentration measured by radioimmunoassay was monitored during pregnancy in 10 females throughout their pregnancy. The breeding season 2004 started on March 18 and ended on May 10, with the peak recorded in April. The duration of first oestrus was 1-12 days. If not mated, the vast majority of females entered second oestrus after 12-55 days. In general, relatively low faecal progesterone values were detected in European mink; an average of 42.69 +/- 4.70 ng/g faeces in oestrous females with a maximum of 176.44 +/- 23.01 ng/g faeces on pregnancy day 12. anova indicated a significant effect of the pregnancy stage. Post hoc comparisons with Fisher least significant difference (LSD) test revealed that faecal progesterone concentrations on days 8 and 12 post coitum (p.c.), but not at the end of pregnancy (day 40), were higher when compared with the initial oestrous level. Implantation in this species occurs on day 12 p.c. and was indicated by prominent uterine swellings and failure to flush the uterine horns beyond this day. Advanced luteogenesis was observed with prominent corpora lutea found in ovaries around the time of implantation. To conclude, European mink is a seasonally polyoestrous species; the early pregnancy of European mink resembles that of European polecat, i.e. in both species, implantation occurs on day 12 p.c. without any implantation delay.

Reproduction of the female ferret (Mustela putorius furo).

The domestic ferret is a seasonally polyoestrous species. Females reach puberty at the age of 8-12 months. Females exhibit a constant oestrus between late March and early August if they are not bred. Increasing tumescence in the pink-coloured vulva is a sign of pro-oestrus. Oestrus can persist for up to 5 months, but once ovulation is induced, either pregnancy or pseudopregnancy ensues. Follicular development and atresia overlap in such a manner that there is a recent cohort of follicles available for ovulation whenever copulation might occur. Copulation may last from 15 min to 3 h, the average being 1 h. Ovulation is induced by pressure on the cervix associated with copulation. After sufficient LH release, the pre-ovulatory follicles mature and an average of 12 oocytes (5-13) per female are ovulated 30-40 h after copulation into the ovarian bursa. The ferret oocytes are most capable of being fertilized up to 12 h after ovulation, i.e. 42-52 h after copulation. Ferret oocytes ovulate at the metaphase of the second meiotic division (MII) embedded in three layers of corona radiata cells. Embryos enter the uterus over a period of several days starting on day 5 after mating. Between days 12 and 13 after mating, the embryos have become implanted in the endometrium. Implantation in the ferret is central with rapid invasion of the uterine epithelium by the trophoblast over a broad area that eventually becomes a zonary band of endotheliochorial placenta. Gestation length is 41 days (39-42 days). The domestic ferret gives birth to an average of eight kits (1-18 kits), which weigh 6-12 g at birth.

Conservation of the European mink (Mustela lutreola): focus on reproduction and reproductive technologies.

The European mink (Mustela lutreola) is a small mammal, which belongs to the Mustelidae family (Carnivora). Earlier, the range of distribution of this species encompassed much of the European continent. During the 20th century, the numbers of European mink declined and the range of its distribution became reduced to three fragmented populations; today this species faces extinction. The urgent necessity for effective conservation efforts to protect the European mink is accepted by the governmental organizations as well as scientific communities of most European countries. In this paper, the reasons for the disappearance of European mink are reviewed and results of past conservation efforts based on captive breeding and reintroduction programmes are critically evaluated in the broad context of modern concepts of conservation genetics and reproductive biology. The data recently obtained on the reproduction and pre-implantation development of European mink and the prospects of incorporation of modern reproductive technologies into the conservation programme of this species are discussed.

Embryo development and embryo transfer in the European mink (Mustela lutreola), an endangered mustelid species.

The European mink is an endangered Mustelidae species and thus requires effective conservation measures, although little is known about reproduction in this species. In particular, preimplantation development has not been studied and, therefore, embryonic development and the growth of embryos was documented in the present study for European mink using light and fluorescent microscopy. Embryos develop in the oviducts and then migrate into the uterus on Day 6 post coitum (p.c.) at the morula stage. Embryos expanded as blastocysts from Day 7 until implantation on Day 12 p.c. Based on these findings, the use of embryo transfer for a conservation programme for the European mink was evaluated. Embryos were flushed from European mink resource females and transferred into the uterine horns of recipient hybrid females (honoriks and nohoriks). These hybrids were obtained by mating European polecat males with European mink females and vice versa. A total of 40 embryos was transferred and 20 live kits were born. The rates of pre- and postnatal survival were 50% and 70%, respectively. Both male and female offspring were lighter at birth in the embryo transfer group compared with naturally born controls, but there was no difference at 3 months of age.

Live cubs born after transfer of OPS vitrified-warmed embryos in the farmed European polecat (Mustela putorius).

The Open Pulled Straw (OPS) method of vitrification has been used successfully for cryopreserving embryos of most domestic animal species. However, there is no report of a successful delivery of offspring after transfer of vitrified embryos in carnivores, even though vitrification has been a successful freezing method for species like swine whose embryos are known to be susceptible to chilling injury. Morulae and blastocysts of farmed European polecat (Mustela putorius) were vitrified and warmed before in vitro culture in modified synthetic oviductal fluid (SOF) for a period from a few hours up to 3 days before being transferred to recipients. Survival rate after vitrification, warming and in vitro culture was 51% (50/98). A total of 50 embryos were transferred surgically into the uteri of four anesthetized recipients. Two recipients delivered a total of eight offspring (2 and 6 each) for an overall survival rate of 16% (eight live cubs/50 transferred embryos). According to our knowledge, these offspring are the first carnivores produced by transfer of in vivo embryos after vitrification by OPS. Based on the present results, we suggest that OPS vitrification can be used as an alternative cryopreservation method for mustelid embryos with pup results comparable to conventional slow freezing.

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius).

Early embryonic development and in vitro culture of in vivo produced embryos in the farmed European polecat (Mustela putorius) was investigated as a part of an ex situ conservation program of the endangered European mink (Mustela lutreola), using the European polecat as a model species. The oestrus cycles of 34 yearling polecat females were monitored by visual examination of the vulval swelling and, to induce ovulation, the females were mated once daily on two consecutive days. Sixteen yearling males were used for mating. The females were humanely killed 3-14 days after the first mating and the uteri and oviducts were collected for embryo recovery. Uterine and oviductal flushings yielded a total number of 295 embryos, representing developmental stages from the 1-cell stage to large expanded and hatched blastocysts. On Day 3 after the first mating, only 1-16-cell stage embryos were recovered. Between Days 4 and 6 after the first mating, 1-16-cell stage embryos and morulae were found. The first blastocysts were recovered on Day 7 after the first mating. The first implanted blastocysts were detected on Day 11 after the first mating. A total number of 85 embryos were in vitro cultured after recovery. Blastocyst production rates for in vitro cultured 1-16-cell stage embryos and for morulae/compact morulae were 68 and 84%, respectively. For all cultured embryos, the hatching rate was 15%. The in vitro culture requirements for the preimplantation embryos of the farmed European polecat remain to be determined before further utilization of the technique.

Human papilloma virus and p53 expression in carcinomas associated with sinonasal papillomas: a Danish Epidemiological study 1980-1998.

OBJECTIVES: To determine a putative role and relation between human papilloma virus (HPV) and p53 in the etiology of sinonasal carcinomas associated with papillomas. STUDY DESIGN: The study group consists of all patients with sinonasal carcinomas associated with papillomas diagnosed in Denmark from 1980 to 1998. After reviewing our national pathological files, tumor tissues from 36 patients were collected, comprising 15% of the total cases of sinonasal carcinomas. In 35 cases a squamous cell carcinoma was demonstrated and in one case an adenocarcinoma was evident. Inverted papilloma was associated with carcinoma in 31 cases and exophytic papillomas in 5 cases. The material was investigated for HPV using polymerase chain reaction analyses with two sets of consensus primers (GP5+/GP6+ and MY09/MY11). The HPV-positive cases were submitted to dot-blot hybridization to establish the HPV type. Using immunohistochemistry, the p53 expression was determined. A p53 overexpression is defined as positive staining in 10% or more of the tumor cells. RESULTS: Among 30 examined cases of carcinomas associated with inverted papillomas, 4 cases were HPV-positive (13%). P53 overexpression was not shown among the HPV-positive cases, whereas p53 overexpression was seen in 21 of the 24 (88%) examined HPV-negative cases. Among the 5 carcinomas associated with exophytic papillomas, HPV was demonstrated together with p53 overexpression in 3 cases (60%). In addition, one case more was with p53 overexpression. CONCLUSION: An inverse relation between HPV and p53 overexpression in sinonasal carcinomas associated with inverted papillomas appears to have been demonstrated. HPV and p53 might also have an etiological role among the carcinomas associated with exophytic papillomas.

Human papillomavirus in conjunctival papilloma.

AIM: To examine conjunctival papillomas for the presence of human papillomavirus (HPV) and koilocytosis. METHODS: Archival paraffin embedded tissue from 55 conjunctival papillomas was analysed for the presence of HPV by polymerase chain reaction and subsequent filter hybridisation. Histological sections of the 55 papillomas were evaluated for the presence of koilocytosis. RESULTS: HPV was present in 48 of 52 (92%) beta globin positive papillomas. HPV type 6/11 were found in 40 of 47 investigated papillomas and a double infection with HPV 6/11 and 16 was identified in a single papilloma. In six papillomas the HPV type could not be identified. Koilocytosis was present in 22 of 55 papillomas (40%). CONCLUSION: There is a strong association between HPV and conjunctival papillomas. HPV type 6/11 is the most common HPV type in conjunctival papilloma. The sensitivity of koilocytosis as an indicator of HPV in conjunctival papilloma is low.

Biophysical and 1H magnetic resonance spectroscopy characteristics of fractionated stallion ejaculates.

The composition of seminal plasma must be determined to assess the possible roles of sex gland secretions in survival of stallion spermatozoa. In the present study, an automated semen collection device and 1H magnetic resonance spectroscopy were used to analyse and compare the composition of seminal plasma from fractionated and nonfractionated stallion ejaculates. The contribution of each semen component to the ejaculate (sequence of production of component and concentration) was evaluated and its relationship to biophysical parameters was determined. 1H magnetic resonance spectroscopy was used to quantify molecules defined as markers of sex gland secretions: carnitine, glycerophosphorylcholine and choline for the epididymides; N-acetyl function of glycoproteins and spermine for the ampullae; acetic acid for the bulbourethral glands; and citric acid for seminal vesicles. The results from 32 ejaculates (four ejaculates from each of four stallions by two collection methods) demonstrated the reliability of the 1H magnetic resonance spectroscopy quantitation, the sequence of sex gland secretion contributions to the ejaculate (bulbourethral glands, epididymides, ampullae and seminal vesicles) and the concomitant appearance of the sperm-rich fraction with secretions from the epididymides and ampullae.

Freezing of stallion semen with addition of glycine betaine.

The effect of addition of glycine betaine to a lactose-EDTA freezing medium on the post-thaw motility of stallion semen was determined. The first three semen-rich fractions of nine stallions were collected with an open-end Krakow artificial vagina on consecutive weekdays. Semen was frozen using the Hannover method with freezing media containing glycine betaine in various concentrations from 0 to 5%. After thawing, sperm motility was analysed both by a light microscope and by a Hamilton-Thorn Motility Analyser. Total and progressive post-thaw motilities of semen containing 0.25-3% glycine betaine did not differ significantly from the total and progressive post-thaw motilities of semen frozen without glycine betaine. The total and progressive post-thaw motilities of semen containing 4 or 5% glycine betaine were significantly lower (P < 0.001) than those of semen without glycine betaine. In conclusion, glycine betaine did not show any beneficial effect on the post-thaw motility of stallion semen when semen was frozen using the Hannover method.