Strong artificial selection in domestic mammals did not result in an increased recombination rate.

Recombination rates vary in intensity and location at the species, individual, sex and chromosome levels. Despite the fundamental biological importance of this process, the selective forces that operate to shape recombination rate and patterns are unclear. Domestication offers a unique opportunity to study the interplay between recombination and selection. In domesticates, intense selection for particular traits is imposed on small populations over many generations, resulting in organisms that differ, sometimes dramatically, in morphology and physiology from their wild ancestor. Although earlier studies suggested increased recombination rate in domesticates, a formal comparison of recombination rates between domestic mammals and their wild congeners was missing. In order to determine broad-scale recombination rate, we used immunolabeling detection of MLH1 foci as crossover markers in spermatocytes in three pairs of closely related wild and domestic species (dog and wolf, goat and ibex, and sheep and mouflon). In the three pairs, and contrary to previous suggestions, our data show that contemporary recombination rate is higher in the wild species. Subsequently, we inferred recombination breakpoints in sequence data for 16 genomic regions in dogs and wolves, each containing a locus associated with a dog phenotype potentially under selection during domestication. No difference in the number and distribution of recombination breakpoints was found between dogs and wolves. We conclude that our data indicate that strong directional selection did not result in changes in recombination in domestic mammals, and that both upper and lower bounds for crossover rates may be tightly regulated.

Single-layer centrifugation separates spermatozoa from diploid cells in epididymal samples from gray wolves, Canis lupus (L.).

Sperm samples may be used for assisted reproductive technologies (e.g., farmed or endangered species) or as a source of haploid DNA or sperm-specific RNA. When ejaculated spermatozoa are not available or are very difficult to obtain, as is the case for most wild endangered species, the epididymides of dead animals (e.g., animals that have been found dead, shot by hunters or poachers, or that that require euthanasia in zoological collections) can be used as a source of sperm. Such epididymal sperm samples are usually contaminated with cellular debris, erythrocytes, leukocytes, and sometimes also bacteria. These contaminants may be sources of reactive oxygen species that damage spermatozoa during freezing or contribute undesired genetic material from diploid cells. We used single-layer centrifugation through a colloid formulation, Androcoll-C, to successfully separate wolf epididymal spermatozoa from contaminating cells and cellular debris in epididymal samples harvested from carcasses. Such a procedure may potentially be applied to epididymal sperm samples from other species.

A survey of dystocia in the Boxer breed.

BACKGROUND: Dystocia occurs more commonly in some breeds of dogs than others. The Boxer breed is one of the highrisk breeds for whelping problems. The aim of this study was to document some reproductive parameters and the frequency of dystocia in Boxers. METHODS: Two questionnaires were sent to the breeders of Boxers in Sweden during 1994 to 1997. Data from 253 whelpings and 1671 pups was received, which constitutes 56.5% of all Boxer litters registered with the Swedish Kennel Club during these years. Data was analysed using Chi-square test, and Fischer's exact test. RESULTS: Dystocia occurred in 32% of the individual bitches, and in 27.7% of all the whelpings. Caesarian section was performed in 22.8% of all the whelpings and in 80.1% of the cases of dystocia. Medical treatment was tried in 20 cases but was successful only in 5 (25%). The dystocia was of maternal origin in 68.6% and of fetal origin in 28.6% of cases. The most common reasons for dystocia were primary uterine inertia (60%) and malpresentation of the fetus (26%). Dystocia increased with increasing age of the bitch from four years of age. Average litter size was 6.6 (+/- 2.2) pups born, and 5.0 (+/- 2.1) pups registered. Pup mortality was 24%. Stillbirths accounted for 6.1% of the pup deaths and 1% died in the neonatal period, while 15.6% of the pups were euthanised, the majority because they had disqualifying white coat colour. Cryptorchidism was observed in 9.8% of the male pups born and in 13.4% of the male pups that were registered. CONCLUSION: The Boxer suffers a high frequency of dystocia, mainly due to uterine inertia, but also fetal malpresentation. Breeders should be advised to include easy whelpings in their breeding program.

The effect of ACTH stimulation on cortisol and progesterone concentrations in intact and ovariohysterectomized domestic cats.

The objective was to evaluate the adrenocortical capacity for cortisol and progesterone production in female cats, both while intact and after ovariohysterectomy. Five privately owned female cats, 1-3 years old, were used in two trials while intact at an inactive stage of the cycle, and again in two trials, 2 weeks after ovariohysterectomy. The four trials were: intact saline injection control trial; intact ACTH injection (0.125 mg); ovariohysterectomized saline injection control trial; and ovariohysterectomized ACTH injection. Blood samples were obtained by an indwelling cephalic vein catheter at -30 and 0 min (immediately before injections) and at 60, 90, 120 and 180 min after injection. The mean basal pre-treatment concentrations of cortisol in the intact and ovariohysterectomized cats were 33 +/- 19 and 32 +/- 19 nmol/L, respectively; the corresponding values for progesterone were 1.1 +/- 0.6 and 0.7 +/- 0.6 nmol/L, respectively. Saline did not alter the serum cortisol or progesterone concentrations. In contrast, both cortisol and progesterone were elevated after ACTH, with peak values at 90 min and returned to basal levels at approximately 180 min. There was a positive correlation between cortisol and progesterone concentrations (r = 0.8, P < 0.05). In some instances, the procedure used to restrain the cats during blood collection induced increases in cortisol and progesterone of the same magnitude as when the ACTH was administered; these effects of restraint could alter the results of assisted reproduction efforts.

Freezing of stored, chilled dog spermatozoa.

The aims of this study were to find out if dog spermatozoa can be stored chilled for 1 or 2 days prior to freezing without a deterioration in post-thaw vitality and longevity, and to compare two extenders; the Uppsala Equex-2 (UE-2) and a TRIS egg yolk extender (EYT). Pooled dog semen was frozen immediately after collection, or was extended and stored at 4 degrees C for 1 or 2 days before freezing. Sperm motility and acrosome integrity were evaluated before freezing and for 6h post thaw at 38 degrees C, while sperm plasma membrane integrity was evaluated post thaw. There were no effects of pre-freeze storage time or extender on post-thaw motility or plasma membrane integrity, but a significant effect of extender (P < 0.0153) on post-thaw acrosomal integrity was found, UE-2 being better than EYT. There was a significant (P < 0.0001) negative effect of post-thaw storage time on acrosome integrity, but this was not influenced by pre-freeze storage time or extender. In conclusion, we found that dog spermatozoa can be frozen after 1 or 2 days of cold storage without significant deterioration in post-thaw motility, acrosome integrity or sperm plasma membrane integrity compared to when frozen immediately after collection. The UE-2 extender was superior to the EYT extender for freezing of cold stored dog spermatozoa.

Hysterographic appearance and uterine histology at different stages of the reproductive cycle and after progestagen treatment in the domestic cat.

The aims of this study were to characterize the hysterographic and histological features of the uteri and to perform immunohistochemistry with proliferating cell nuclear antigen (PCNA) in the cat endometrium at various stages of the reproductive cycle and after treatment with exogenous progestagen. Seventy-four female domestic cats submitted for routine ovariohysterectomy were categorized into six groups: inactive (n=20), follicular (n=9), luteal (n=18), and postpartum (n=12) stages of the reproductive cycle; cats given medroxyprogesterone acetate for estrus prevention (MPA group) (n=12); and cats with uterine pathological lesions (n=3). Hysterography was performed and the relation of the uterine and luminal shape in the hysterogram with the stage of the reproductive cycle as well as with any pathological conditions of the uterus was evaluated. The uteri and ovaries were thereafter surgically removed and sectioned for histological examination. The PCNA was used to demonstrate the expression of endometrial epithelial cell growth. The hysterographic appearance was found to differ between the six groups of cats. A straight uterine cavity was characteristic for cats in the inactive stage, whereas a wavy uterine cavity was characteristic for cats in the follicular stage. In the luteal stage, the luminal cavity of the uteri differed in shape with increasing progesterone concentration from straight to irregular wavy or coiled. The coil shaped uterine lumen seen in the MPA treated and pathological groups was considered also to be an expression of a progestagenic effect. Waviness and coiling of the uterine lumen was related to a proliferation of the endometrial glands, whereas irregular filling defects were indicative of endometrial cystic changes. This study is the first to demonstrate the expression of PCNA in the cat endometrium although no differences were found between the six groups of cats. The hysterographic appearance was found to differ according to stage of the reproductive cycle and pathological conditions. Thus, a normative hysterogram is now available for diagnosing the reproductive stage and uterine changes in cats developing endometrial hyperplasia with and without cystic changes.

Influence of glucose and fructose in the extender during long-term storage of chilled canine semen.

The use of chilled, extended semen in dog breeding is becoming increasingly popular as preparation and transportation is less expensive and regulations are often less complicated than for frozen semen. Sugar is one of the main constituents in semen extenders, and glucose and fructose are metabolized in separate pathways by freshly ejaculated dog sperm. In this study, glucose, fructose or an equal mixture of both were used in an egg-yolk-tris (EYT) extender at two different concentrations (10 and 70 mM). EYT extender without sugar supplementation, providing only the glucose (3-4 mM) originating from the egg-yolk, served as a control. The longevity of the chilled semen at 5 degrees C was 23 days: the quality of physical and functional characteristics decreasing with time. Glucose and fructose had a strong influence on motility and movement patterns of chilled canine semen. The beneficial effect of 70 mM sugar concentrations compared to 10 mM and the control was pronounced, and maintained sperm motility > or = 70% for 8 days of storage, compared to for 4 days in the control extender. Fructose maintained higher sperm motility than did glucose and the mixture. VAP values were higher in sugar-supplemented extenders (P < 0.05). Neither type nor concentration of the two sugars influenced sperm plasma membrane, acrosome integrity or the acrosome reaction following ionophore challenge (ARIC). Sugar consumption by dog sperm varied between the different periods of storage and with sugar concentrations provided in the extenders. Glucose consumption by dog sperm was greater than fructose consumption when both sugars were present in equal amounts, indicating that dog sperm used glucose in preference to fructose. In conclusion, the major influence of the two sugars on chilled semen was to support motility. EYT extender supplemented with fructose at a concentration of 70 mM was found to be the best of the tested extenders for long-term preservation of chilled canine semen.

Distribution of spermatozoa in the female reproductive tract of the domestic cat in relation to ovulation induced by natural mating.

The purposes of this study were to demonstrate the localization of spermatozoa in the reproductive tract of female domestic cats before (30 min and 3 h after mating) and after ovulation (48 and 96 h after mating), and to evaluate the efficiency of two techniques for studying sperm distribution. Estrus was induced in twenty-four female cats using 100 IU eCG and the females were divided into four groups with six females per group. The same male cat was used for mating with all the females. One group of six females was mated once; the others were mated four times in 1 h. Ovariohysterectomy was performed at 30 min, 3 h, 48 h, and 96 h after mating and the excised reproductive tracts were divided into seven segments on each side: infundibulum, ampulla, isthmus, uterotubal junction (UTJ), cranial and caudal uterine horn, and uterine body. The vagina and the lumina of the segments from one side were flushed with 0.5 ml PBS. The flushed and the non-flushed segments from the contralateral side were then fixed in 3% neutral buffered formalin and processed for routine histology. The numbers of spermatozoa in the flushings and in 40 histological sections from each segment were counted. Before ovulation, the majority of spermatozoa was detected in the vagina and the uterine segments, whereas after ovulation, significantly higher numbers of spermatozoa were present in the uterine tubal segments. The decreasing gradient in sperm numbers at 30 min and 3 h after mating between the vagina, the uterine segments, including the UTJ, and the uterine tubal segments indicated that the cervix and the UTJ served as barriers for sperm transport in the cat. The UTJ and the uterine crypts acted as sperm reservoirs before ovulation whereas the isthmus was a sperm reservoir around the time of ovulation. There was no difference in sperm numbers in the tissue sections between flushed and non-flushed segments, implying that the flushing technique only recovered some intraluminal spermatozoa while most of the spermatozoa remained in the epithelial crypts. This was further supported by the finding that significantly higher numbers of spermatozoa were recovered in the flushings at 30 min and 3 h after mating, when more spermatozoa were free in the lumina, than at 48 and 96 h after mating, when the majority of the spermatozoa were entrapped in the uterine epithelial crypts.

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa.

Cryopreservation of epididymal spermatozoa is a potentially valuable tool for preserving genetic material from individuals of endangered species that die accidentally. Improvement of sperm-freezing protocols would increase the efficacy of gene banking from endangered felids, and the domestic cat can be used as a model for the wild felids. Addition of the detergent Equex STM paste to semen freezing extenders has been found to improve post-thaw survival and longevity of spermatozoa from various species but has never been tested for cat spermatozoa. Spermatozoa from cats with a high percentage of morphologically abnormal spermatozoa are more susceptible for cold injury and osmotic stress than spermatozoa from normozoospermic cats. Therefore, the aims of this study were to investigate: (a) if addition of Equex STM paste to a semen freezing extender would improve post-thaw sperm survival, and (b) if there is a relation between the percentage of morphologically normal spermatozoa and cryopreservation induced damage in cat epididymal spermatozoa. Spermatozoa harvested from epididymides of 10 male cats were frozen in a Tris egg yolk extender with or without the addition of Equex STM paste (0.5%, v/v). Sperm motility, membrane integrity and acrosomal status were evaluated immediately after harvesting, and at 0, 2, 4 and 6 h post-thaw. Sperm membrane integrity and acrosomal status were also evaluated after cooling to 4 degrees C, just before freezing. Cooling did not cause significant damage to the spermatozoa, whereas freezing damaged sperm membranes and acrosomes. Addition of Equex to the freezing extender had a significant positive effect on the percentage of intact acrosomes immediately after thawing (P > 0.05), but had a negative effect on the longevity of the spermatozoa; the percentages of membrane intact and motile spermatozoa being significantly lower in the presence of Equex than in the controls at 6h after thawing. The percentage of morphologically normal spermatozoa was not found to be correlated with either cryopreservation induced acrosome or plasma membrane damage, or with post-thaw motility (P > 0.05). The results clearly show that addition of Equex STM paste in the freezing extender protects the acrosomes of cat epididymal spermatozoa during the freezing--thawing process, but reduces the sperm longevity during in vitro incubation at 38 degrees C. Our results also indicate that the percentage of morphologically normal epididymal spermatozoa is not correlated with cryopreservation induced sperm damage using the described freezing protocol.

Efficacy of the anticaspase agent zVAD-fmk on post-thaw viability of canine spermatozoa.

Cryopreservation protocols for gametes are constantly improved with the aim of increasing the post-thaw viability of gametes. It is becoming clear that stress, resulting from cryopreservation, reduces cell numbers by apoptosis. Apoptosis, or programmed cell death, is a gene-activated event that occurs as a normal consequence of development and as a result of cellular stress. Apoptosis is mediated by the family of cysteine-dependent asparate-specific proteases (caspases). The present study was designed to test the hypothesis that addition of an anticaspase (zVAD-fmk) that has inhibitory properties against caspases and apoptosis to semen extenders and to the thaw medium would increase post-thaw viability of canine spermatozoa. Extenders were added in a two-step process. A dose of 100 microM caspase inhibitor was used. Four groups (n=6 for each) were composed based on the presence or absence of the caspase inhibitor: Group I (control), no caspase inhibitor in the extender or the thaw medium; Group II, caspase inhibitor in the thaw medium; Group III, caspase inhibitor in Extender II; and Group IV, caspase inhibitor in both Extender II and the thaw medium. Post-thaw motility, plasma membrane integrity, and acrosome status were investigated. The addition of caspase inhibitor to Extender II or to the thaw medium failed to improve the parameters that were studied. The results suggest that this caspase inhibitor may not be beneficial to the post-thaw motility of canine spermatozoa if used at this concentration.

Creatine phosphokinase in domestic cat epididymal spermatozoa.

Mammalian spermatozoa that have not completed final testicular sperm maturation have residual cytoplasm and increased creatine phosphokinase (CK) content. This study determined: (1) if CK could be detected by immunostaining cat spermatozoa from the caput, corpus, and cauda epididymis, (2) fluctuations in the proportions of spermatozoa with mature or immature CK-staining patterns during epididymal sperm transit, and (3) how well sperm maturity (as determined by a CK marker) correlated with testicular or epididymal dysfunctions associated with morphological sperm abnormalities. One epididymis was collected from each of 37 cats after orchiectomy and processed immediately to allow sperm morphology evaluations on a 'regional' basis. Sperm released from the contralateral epididymis were evaluated for motility, sperm membrane integrity, and immunostaining with CK-B antibodies. Proportions of spermatozoa with malformed or detached heads, proximal droplets and acrosomal or midpiece abnormalities decreased (P < 0.05) from the caput to the cauda epididymis. In contrast, proportions of spermatozoa that were motile, membrane-intact or with flagellar abnormalities or distal droplets increased (P < 0.05) from the caput to cauda region. Percentages of spermatozoa with an immature CK-staining pattern also decreased (P < 0.05) with epididymal transit (which differs from that reported for the human and stallion). There was no correlation (P > 0.05) between sperm morphology and the CK-staining patterns. In summary, the results reveal that some specific sperm malformations in the domestic cat are of testicular origin, whereas others develop during epididymal transit.