Reproductive patterns in the domestic dog--a retrospective study of the Drever breed.

The aim of this study was to examine the differences, between seasons of the year, in the distribution of matings and whelpings, litter size, pup deaths, and sex ratio in domestic dogs. Furthermore, we wanted to examine the effects of age and parity of the bitch at the time of whelping on litter size, as well as the effect of litter size on gestational length. A final aim was to investigate the fertility and frequency of whelping problems in a private kennel of Drever dogs. Data from the Swedish Kennel Club (SKK) registry for the Drever breed during 1995-2006, comprising a total of 2717 litters, were analyzed together with more detailed data from a private, professional kennel of Drevers, with a total of 285 matings and 224 whelpings, during the same time period. The most matings took place during winter, and the fewest during summer; consequently, most whelpings occurred during the winter and spring seasons. Of the 285 mated bitches, 78.6% whelped, 6.25% experienced dystocia, and 5.36% underwent Cesarean section. The pup death rate was 7.6%. The largest litters were born during spring. Litter size was negatively correlated with duration of pregnancy (r=-0.18). Each pup more than average caused a shortening of the gestation by 0.25 days, and each pup less a corresponding lengthening. Bitches giving birth to their first litter after 4 years of age produced a smaller litter than younger bitches. Litter size decreased after 5 years in all bitches. The number of born pups at the private kennel increased from the first to the third parity, then decreased. The number of registered pups increased from the first to the second parity in the SKK data and from the second to the third parity in the data from the private kennel, then decreased. Mating a bitch only once resulted in a smaller litter size. None of the studied factors had any effect on the sex ratio of the pups. There were significant differences between males in whelping rate among the mated bitches, but no difference in mean litter size, which indicates a female problem rather than a male one. Available data suggest that the domestic dog is still under considerable seasonal influence, although modified by ambient and management factors.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen.

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.

Induced immotility during long-term storage at +5 degrees C does not prolong survival of dog spermatozoa.

This study investigated whether the immotility induced by the CLONE chilled semen extender prolongs the lifespan of dog spermatozoa stored at 5 degrees C, compared with a Tris-egg yolk-glucose (TG) extender, which maintains motility. Pooled semen was split in four aliquots, centrifuged, and the four sperm pellets mixed with TG extender; with the CLONE chilled semen (CL) extender; with TG extender mixed with an activator (TG+A(TG)); or with the CLONE extender mixed with the CLONE activator (CL+A(CL)). Samples were stored at 5 degrees C for 23 days and examined 12 times for sperm motility, plasma membrane and acrosome integrity, glucose consumption, and DNA fragmentation index (DFI). The experiment was performed in triplicate. Glucose consumption was not significantly different between extenders until the period 15-23 days, when it was higher in CL and CL+A(CL) than in TG (P=0.0055) and TG+A(TG) (P=0.0010). No breakdown of DNA chromatin (P>0.05) occurred until day 14. Spermatozoa preserved in TG or TG+A(TG) showed better values for all the different parameters throughout the experiment compared with sperm subjected to CL or CL+A(CL). In conclusion, the immotility induced by the CLONE chilled semen extender during long-term cold storage at 5 degrees C did not prolong the lifespan of spermatozoa compared with the lifespan following storage in Tris-egg yolk-glucose. In addition, our results indicate that good quality dog semen may possibly be stored for up to 14 days in TG extender at 5 degrees C, with retained fertilizing capacity. In vivo studies should, however, be performed to further support this conclusion.

Sperm morphology in the domestic cat, and its relation with fertility: a retrospective study.

Knowledge about normal ranges in semen quality and the association between sperm morphology and fertility in felids is limited. The aims of this retrospective study were to (1) define a normal spermiogram in cats; (2) evaluate possible effects of season, age and breed on sperm morphology; and (3) evaluate the relationship between sperm morphology and fertility. Semen samples collected by electroejaculation from 52 cats were evaluated for sperm morphology. The cats constituted two groups: a general population of cats (n = 48) and cats examined because of poor breeding records (n = 4). The general population was divided into household (n = 20), pedigree (n = 19) and colony cats (n = 9) and into three age classes, <12 months, 12-59 months and >or=60 months. The median percentage of normal spermatozoa in the general population was 44.0% (range 1.0-91.0%). Criteria were tentatively set for what was considered a normal spermiogram. The mean percentage of normal spermatozoa was higher during February to July than during August to January (p < 0.05). Pedigree cats had a lower mean percentage of normal spermatozoa than did household cats (p < 0.05). Age had no effect on the percentage of normal spermatozoa but was positively correlated with the percentage of proximal droplets. Of the cats with <40% normal spermatozoa (n = 19), all those with known breeding records (n = 11) had produced litters. The four cats examined because of poor breeding results had higher percentages of different sperm abnormalities than tentatively stipulated for the normal spermiogram. In two of these cats both sperm morphology and fertility changed over time.

Reproductive patterns and reproductive pathologies of stray bitches in the tropics.

The objectives of this study were to determine the annual reproductive pattern and to estimate the frequency of reproductive pathologies in female mongrel stray dogs under tropical conditions. The genital tracts of 300 mongrel bitches from a municipal dog pound were examined post-mortem from January to December 2003. Season of the year, age, size, and body condition score (BCS) were recorded for each dog. The year was divided into three seasons: warm-dry (March-June), warm-humid (July-October), and fresh-humid (November-February). Distribution of estrus periods was not influenced by any of the factors studied (i.e. season, age, size, BCS). A significantly lower number of pregnancies were recorded during the warm-dry season, probably as a consequence of embryo resorption. Underweight animals had a significantly lower percentage of pregnancies than bitches of ideal BCS. More ovulations per bitch occurred during the warm-humid season than during the other seasons of the year, probably due to climatic factors. Bitches of medium and large size had more ovulations than those of small size. Of the 300 bitches examined, 43.5% had one or more genital pathologies. The most frequent pathologies found in the ovary, uterus, and vagina were epoöphoron cysts (6.7%), serosal inclusion cysts (5.0%), and transmissible venereal tumors (15.3%), but the capacity of the females to come into estrus or to become pregnant was not affected by these conditions. We concluded that stray domestic bitches in the tropics were not seasonal breeders, but their reproductive pattern was apparently modified by environmental factors such as temperature and probably photoperiod. Although several bitches in the present study had reproductive pathology, the most prevalent pathologies did not adversely reproductive capacity.

Pathological conditions of the reproductive organs of male stray dogs in the tropics: prevalence, risk factors, morphological findings and testosterone concentrations.

The objective of this study was to estimate the prevalence of and risk factors for pathological conditions of the reproductive organs in stray dogs under tropical conditions. Three hundred and eighteen dogs were examined post-mortem in the period from 1 July 2002 to 30 June 2003. Before killing, a blood sample (from the cephalic vein) for testosterone assay was taken. Pathological conditions of the reproductive organs were found in 135 of the dogs (42.5%) and in 175 of the testes (64.8%). The most frequent pathologies found were testicular degeneration, cryptorchidism, testicular hypoplasia and testicular tumours (in 15.1%, 6.6%, 6.6% and 5.4% of the dogs and 15.1, 4.6, 6.0 and 3.5 of the testes, respectively). Transmissible venereal tumour (TVT) was seen in 5.4% of the dogs. Testicular degeneration was more common in old dogs and underweight dogs (p < 0.05). Testicular tumours were 14.3 times more common in cryptorchid dogs. Age was another important factor for the development of testicular tumours (p < 0.05). Lower levels of testosterone concentration (p < 0.05) were observed in dogs with advanced testicular degeneration (0.7 +/- 0.8 nM), dogs with hypoplastic testicles (0.8 +/- 0.9 nM) and dogs with one degenerated and one retained testis or with bilateral cryptorchidism (1.2 +/- 0.9 nM) compared to dogs with one or two normal testes (7.0 +/- 5.5 nM). Testicular volume and weight were significantly lower in degenerated, hypoplastic and retained testes compared with the contralateral normal testis. Some spermatogenic activity was found in three of the retained testes, producing oligozoospermic smears with a high percentage of sperm abnormalities. No comparable epidemiological data about male pathological conditions of the reproductive organs in the dog is available. The prevalence found in this study, yet, appears high.

Reproductive patterns of stray male dogs in the tropics.

Two studies were performed to determine annual reproductive patterns in stray male dogs in the tropics. In Study 1, four dogs housed individually outdoors were monitored once monthly for 12 months, including collection and assessment of semen, measurements of scrotal width, and determination of serum testosterone and prolactin concentrations. In Study 2 (conducted concurrently), a single blood sample (for serum testosterone concentration) was collected from 220 clinically healthy dogs, and after euthanasia, scrotal width and morphology of epididymal sperm were determined. The year was divided into three seasons: warm-dry (March to June); warm-humid (July to October) and fresh-humid (November to February). In Study 1, scrotal width, ejaculate volume, sperm count and motility were significantly lower during the fresh-humid season and sperm midpiece abnormalities were significantly more common during the warm-humid and fresh-humid seasons. Serum testosterone concentrations remained constant during the year. Prolactin concentrations did not differ significantly among seasons, but had a well-defined increase from the beginning of March to the end of August. In Study 2, sperm morphology was similar to in Study 1 and serum testosterone concentrations varied nonsignificantly during the year. Environmental factors, e.g. daylength may have influenced circannual changes in prolactin secretion. Seasonal variations in some reproductive tract and seminal traits were significant but of small magnitude and the percentage of morphologically normal sperm did not vary significantly among seasons. In conclusion, healthy male dogs constantly produced sperm and were apparently fertile throughout the year.

Evaluation of a Burdizzo castrator for neutering of dogs.

A Burdizzo castrator was evaluated for the neutering of dogs. Histological and morphological changes of spermatic cells and peripheral serum testosterone after challenge with a GnRH-analogue (gonadorelin) were assessed. There was a control group (G1), a surgically castrated group (G2) and a Burdizzo group (G3) divided in two, G3a receiving two crunches in each spermatic cord and G3b receiving one crunch in each spermatic cord. Sixteen days after application of the Burdizzo blood samples were taken from the dogs at 30 min interval during 2 h; after the second sample the dogs were treated with 1 mug/kg body weight of gonadorelin i.v. The same protocol of gonadorelin challenge was performed in G1 and G2 dogs. The G2 dogs were surgically castrated after the second blood sample, before the gonadorelin treatment, and the G1 dogs after the last blood sample. The excised gonads were examined histologically, and sperm smears were prepared from the caudae epididymidis. The testes and plexus pampiniformis of the G1 and G2 dogs had a normal histological appearance, and they had morphologically normal epididymal sperm cells. In all G3 dogs, there was an acute fibrosis with an inflammatory reaction in the plexus pampiniformis. The testes from the G3a dogs showed diffuse areas of infarction and degeneration of the parenchyma. Similar but less diffuse lesions were seen in group 3b dogs. The deferent ducts from all G3 dogs showed vasitis and/or sperm granulomas. Azoospermia or sperm malformations were observed in the epididymal smears from the G3 dogs. Testosterone concentration in the G1 dogs increased after gonadorelin application (p < 0.0001). The G2 dogs had basal testosterone levels after castration (p < 0.001) and did not respond to gonadorelin. Groups 3a and b showed a slight but non-significant increase in testosterone concentration after gonadorelin challenge, supposedly due to the reduction of testicular blood flow and loss of testicular interstitial tissue.

Investigation of cervical patency and uterine appearance in domestic cats by fluoroscopy and scintigraphy.

The cervical patency of six domestic female cats was monitored under sedation by infusion of contrast medium (Omnipaque) into the cranial vagina during early oestrus, mid-oestrus, late oestrus and interoestrus or a radiopharmaceutical ((99m)Tc-HSA) during mid- and interoestrus in a non-ovulatory oestrous cycle. The transport of the contrast medium or the radiopharmaceutical through the cervix and within the uterine horns was observed under fluoroscopy and with the aid of scintigraphy. In three of the queens, transcervical transport of contrast medium was demonstrated in all stages of oestrus, in one queen during mid-oestrus, late oestrus and 1 day after oestrus, and in two queens only during late oestrus. The relations between the cervical patency to the contrast medium and the oestrous behaviour, cornification of the vaginal cells and the serum oestradiol-17beta concentration were evaluated, and a relationship was found between the cervical patency and the degree of vaginal cornification. Transcervical transport of the radiopharmaceutical was observed in three queens during mid-oestrus. When the cervix was open, hysterography under a fluoroscope and hysteroscintigraphy were performed. The fluoroscopic and scintigraphic recordings revealed the patterns of the uterine contractions during oestrus in both ascending and descending directions, and the movement of the uterine contents back and forth between the uterine horns. The hysterograms were classified according to the shape of the uterine horns and the appearance of the endometrial lining. Spiral-shaped uterine horns with a smooth inner contour were observed in two queens, and a corkscrew appearance with irregular filling defects in the uterine lumen was shown in two queens that had developed subclinical cystic endometrial hyperplasia. These findings demonstrated that fluids or particles deposited in the cranial vagina of the cat can be transported into the uterus during some stages of the oestrous cycle. The fluoroscopic and scintigraphic techniques developed in this study may be further modified to permit more detailed studies of uterine contractile patterns and sperm transport in the feline female reproductive tract. Hysterography proved useful to diagnose uterine disease. The information on cervical patency is of value also for the development of techniques for artificial insemination in this species, and should be studied also in the ovulatory cycle.

Zona pellucida binding assay--a method for evaluation of canine spermatozoa.

The present study describes the use of a zona pellucida binding assay for the evaluation of canine spermatozoa. A zona pellucida binding assay is a sperm evaluation test that is practical to perform and provides potentially useful information on the damage caused to spermatozoa by new methods of sperm storage. The addition of the detergent Equex STM paste to the cryopreservation extender has a positive effect on the zona pellucida binding capacity of cryopreserved spermatozoa. A large number of sperm-oocyte complexes need to be evaluated because of the variability in sperm binding capacity among oocytes. However, this does not constitute a major problem as canine oocytes can be stored before use in a zona pellucida binding assay and sperm-oocyte complexes can be fixed and stored until evaluation.

Transcervical catheterization and cervical patency during the oestrous cycle in domestic cats.

The aims of the present study were to develop a device for vaginal and transcervical catheterization in domestic cats, and to study cervical patency during the various stages of the oestrous cycle. Seventeen queens submitted for routine spaying were included in the study. A vaginal catheter was designed from a urinary catheter for dogs, to fit into the ventral vaginal fornix, and a 3.5 French tomcat catheter was used as an inner transcervical catheter. Cervical patency was studied by infusing 0.5 ml Urografin into the cranial vagina and taking X-rays of the queens after 5 min. The Urografin did not enter the uterus, even in the oestrous queens. Transcervical catheterization was then attempted. The correct placement of the intrauterine catheter was confirmed by injecting green food colour mixed with penicillin G and observing the presence of stain in the uterine horns during surgery. Catheterization was successful in 13 of 17 queens: six of nine in interoestrus, three of three in oestrus, one of two in metoestrus and three of three in the postpartum period. Transcervical catheterization is a non-invasive technique that is likely to improve the success rate of assisted feline reproduction, and is potentially a useful non-surgical technique for diagnosis and therapy of uterine diseases.

Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation.

The aims of the present study were: (i) to validate the accuracy of flow cytometry for assessment of viability and acrosomal status of canine spermatozoa; and (ii) to evaluate the cryopreservation protocols currently used for dog spermatozoa using flow cytometry. Data obtained by flow cytometry analysis of fresh dog spermatozoa stained with carboxyfluorescein diacetate (CFDA) and propidium iodide, or with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) and propidium iodide, were compared with those obtained by microscopic evaluation. The results demonstrated that flow cytometry is a precise method for evaluating the viability and acrosomal status of fresh samples of dog semen. A new triple staining procedure, using carboxy-SNARF-1, propidium iodide and FITC-PSA, was developed and was an efficient method for evaluating the following aspects of cryopreservation protocols for dog spermatozoa: (i) addition of 0.5% (v/v) Equex STM paste to a Tris-egg yolk-based extender; (ii) dilution of the semen in one or two steps; (iii) freezing semen by placing 0.5 ml straws horizontally above liquid nitrogen in a styrofoam box or lowering them vertically into a liquid nitrogen tank; (iv) thawing semen at two different rates; (v) packaging semen at different sperm concentrations; and (vi) diluting semen at different rates after thawing. The highest sperm survival and longevity was obtained when Equex was present in the semen extender, the semen dilution was performed in two steps to obtain a concentration of 2.0 x 10(8) spermatozoa ml-1, the freezing was carried out using the styrofoam box, the straws were thawed at 70 degrees C for 8 s and the semen was diluted 1:4 after thawing.

Prediction of the oocyte recovery rate in the bitch.

In order to investigate the possibility of predicting the recovery rate of oocytes for use in a sperm-zona pellucida binding assay, ovaries were obtained from 67 bitches of 37 different breeds, and cumulus-oocyte complexes (COCs) were recovered by mincing the ovaries with a scalpel. The mean number of COCs recovered was 37.2 +/- 34.1 (range 0-145) per ovary. Age significantly affected COC recovery rates. From bitches 1-6 years old, 54.2 +/- 35.1 COCs/ovary were recovered, compared to 26.4 +/- 29.0 from bitches 7-13 years old (P = 0.003). The morphology of the uterus or the presence or absence of ovarian structures had no significant effect on COC recovery rates, although there was a tendency for more COCs to be recovered from ovaries with only follicles visible on the surface. There were no significant correlations between body weight or ovarian weight and COC recovery rates. There was a high correlation in the COC recovery rate between the two ovaries of a bitch, enabling an approximate estimation of the COC recovery rate from the second ovary when the COCs from the first ovary have been recovered. The large variation in COC recovery rates between bitches stresses the need for storage of canine oocytes in order to secure a high enough number of oocytes for a homologous sperm-zona pellucida binding assay in the dog.

Effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa.

The objectives of this study were to evaluate the effects and interactions of freezing dog semen using 4 different sperm concentrations (50 x 10(6), 100 x 10(6), 200 x 10(6) and 400 x 10(6) spermatozoa/mL) in 0.5-mL straws and diluting the thawed semen at 4 different rates (1:0, 1:1, 1:2 and 1:4) on post-thaw survival and longevity of dog spermatozoa during incubation at 38 degrees C. Fifteen ejaculates were collected from 12 dogs and pooled. The semen pool was divided into 4 aliquots containing respectively 4,200 x 10(6), 2,100 x 10(6), 1,050 x 10(6) and 525 x 10(6) spermatozoa, which were centrifuged. Sperm pellets were rediluted with TRIS-glucose-egg yolk extender containing 5% glycerol and 0.5% of Equex STM Paste to obtain the designated sperm concentrations. The semen was frozen in 0.5-mL straws 4 cm above liquid nitrogen (LN2). The straws were thawed at 70 degrees C for 8 sec and the contents of each straw were divided into 4 aliquots and diluted with TRIS buffer at 38 degrees C at rates of 1:0, 1:1, 1:2 and 1:4 (semen:buffer), respectively, making a total of 16 treatments. Sperm motility was subjectively evaluated after thawing and at 1-h intervals during 8 h of incubation at 38 degrees C. Plasma membrane integrity and acrosomal status were evaluated at 1, 3, 6, 12 and 18 h post-thaw using a triple-staining procedure and flow cytometry. For data pooled across the post-thaw dilution rate, motility was higher (P< 0.001) in samples frozen with 200 x 10(6) spermatozoa/mu. The integrity of sperm plasma membranes after 18 h incubation was higher (P<0.05) in samples frozen with 200 x 10(6) and 400 x 10(6) spermatozoa/mL. For data pooled across sperm concentration, samples diluted at a rate of 1:2 or 1:4 had better (P<0.001) motilities after 8 h of incubation than undiluted samples or those diluted at 1:1. The integrity of the sperm plasma membranes was higher (P<0.001) at increasing dilution rates. When the 16 treatments were compared, the best longevity was obtained when semen packaged at a concentration of 200 x 10(6) spermatozoa/mL was diluted immediately after thawing at 1:4 dilution rate.

Effects of Equex, one- or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa.

The objectives of the present study were to evaluate the effects of adding Equex to a TRIS-extender, diluting the semen in 1 or 2 steps, freezing according to 2 methods, thawing at 2 rates, and the interactions between these treatments, on the post-thaw survival of dog spermatozoa at 38 degrees C. Ten ejaculates were obtained from 8 dogs. Each ejaculate was centrifuged, and the seminal plasma was discarded. Each sperm pellet was diluted with 2 mL of a TRIS-glucose-egg yolk extender containing 3% glycerol (Extender 1 [Ext-1]). Ejaculates were then pooled (9 x 10(9) spermatozoa), and Ext-1 was added to obtain 200 x 10(6) spermatozoa/mL. The semen pool was carefully mixed and divided into aliquots, and processed according to a 2 x 2 x 2 x 2 factorial design to evaluate the effects of 1) adding the same volume of a second TRIS-glucose-egg yolk extender with 7% glycerol that contained (Ext-2-E) or didn't contain (Ext-2) 1% of Equex STM Paste (final concentration of spermatozoa 100 x 10(6) spermatozoa/mL, glycerol 5%, Equex 0% [Ext-2] or 0.5% [Ext-2-E]); 2) diluting the semen in 1 step (adding Ext-2 or Ext-2-E before equilibration) or in 2 steps (adding Ext-2 or Ext-2-E after equilibration, just before the freezing operation); 3) freezing the straws horizontally in a styrofoam box 4 cm above liquid nitrogen (LN2) or by lowering them vertically into a LN2 tank in 3 steps; and 4) thawing at 70 degrees C for 8 sec or at 37 degrees C for 15 sec. A total of 16 treatment combinations were evaluated. Sperm motility was evaluated after thawing and at 1-h intervals during 7 h of incubation at 38 degrees C by subjective examination and by using a CASA-system. Plasma membrane integrity and acrosomal status were evaluated simultaneously at 1, 3 and 6 h post-thaw using a triple fluorescent staining procedure and flow cytometry. The best post-thaw survival and thermoresistance of spermatozoa was obtained when Equex was present in the extender (P<0.0001); the semen dilution was performed in 2 steps instead of 1 (P<0.0001); the freezing was carried out using the box instead of the tank (P<0.05); and the straws were thawed at 70 degrees C for 8 sec instead of at 37 degrees C for 15 sec (P<0.0001).

Sperm binding capacity and ultrastructure of the zona pellucida of stored canine oocytes.

Sperm binding to the zona pellucida is a prerequisite for fertilization, and tests that evaluate this function have been described for several species. When carrying out such tests in the canine species, ovaries or oocytes have to be stored to obtain a sufficient number of oocytes at the time of testing. In the present study, the sperm binding capacities of salt-stored oocytes and oocytes from deep frozen ovaries were measured and compared with that of fresh oocytes. Two different procedures for washing the sperm-oocyte complexes (gentle and tough) were used before evaluating the number of bound spermatozoa. The total number of oocytes that bound spermatozoa was significantly lower for both salt-stored and deep frozen oocytes compared with fresh oocytes. Significantly fewer spermatozoa bound to stored oocytes than to fresh oocytes (P

Evaluation of chilled and frozen-thawed canine spermatozoa using a zona pellucida binding assay.

Zona pellucida binding assays provide information about the fertilizing ability of spermatozoa. A zona-binding assay for canine spermatozoa using intact, denuded homologous oocytes has not been evaluated previously. In the present study, an assay using canine oocytes derived from frozen-thawed ovaries was evaluated using three types of semen: fresh untreated; killed; and a 50:50 mixture of untreated and killed spermatozoa. The assays were performed on 3 x 20 oocytes for each sperm treatment, using semen from pooled ejaculates (0.5 x 10(6) spermatozoa in each 50 microliter droplet containing five oocytes). There was a significant difference (P < 0. 001) between all treatments. Thereafter, the same procedure was used to evaluate methods of chilling and freeze-thawing of canine semen. There was a trend (P = 0.067) for more sperm binding after 1 day of chilling compared with after 4 days of chilling. Semen samples frozen using an extender (with or without the addition of Equex STM paste) were evaluated. Equex had a significant (P = 0.034) positive effect on the capacity of the spermatozoa to bind to the zona pellucida. In conclusion, the addition of a zona pellucida binding assay to established in vitro tests should give a better estimate of the damage caused by the various procedures when developing new techniques for chilling and freeze-thawing. Furthermore, the present study showed that chilling for 4 days tended to reduce the zona-binding capacity of the spermatozoon, and that Equex STM paste had a beneficial effect on the capacity of the frozen-thawed spermatozoon to bind to the zona pellucida.

In vitro capacitation of fresh, chilled and frozen-thawed dog spermatozoa assessed by the chloretetracycline assay and changes in motility patterns.

The effect of preservation on capacitation status of dog spermatozoa was investigated. Split ejaculates from six dogs were assessed as fresh, chilled for 24 h and rewarmed, and frozen-thawed samples. Capacitation-like status was assessed using the chlortetracycline (CTC)-assay and the measurement of sperm motility patterns using a computer-assisted sperm analyzer. Evaluations were performed on washed spermatozoa immediately after dilution in a Tris-fructose-citrate buffer (TFC) or in canine capacitation medium (CCM), and at 2-h intervals during 8 h of incubation in 5% CO2 in air, at 37 degrees C. Preservation decreased significantly the proportion of uncapacitated spermatozoa. In TFC, at hour 0, chilled-rewarmed and frozen-thawed samples had a significantly lower proportion of uncapacitated, viable spermatozoa than the fresh samples (P<0.05) according to the CTC-assay. The time course of capacitation was accelerated in the preserved samples, compared to the fresh ones. During incubation in CCM, the mean time from hour 0 to when, according to the CTC-assay, the highest proportion of capacitated spermatozoawas present in the samples (time-to-peak), was 4 h for fresh and 2 h for chilled-rewarmed and frozen-thawed samples (P<0.1). The highest values for curvilinear line velocity (VCL) and lateral head displacement (LHD), thought to be descriptive of sperm hyperactivation, were also observed 4 and 2 h after incubation began, in the fresh and the preserved samples, respectively. The difference in time-to-peak for VCL and LHD between fresh, chilled-rewarmed and frozen-thawed semen samples was statistically significant (P<0.02). It can be concluded that based on the CTC-assay and the analysis of motility patterns, capacitation-like changes in dog semen seem to be both initiated and accelerated by the preservation procedures.

Radiographic pelvimetry for assessment of dystocia in bitches: a clinical study in two terrier breeds.

Radiographic pelvimetry was used to assess the role of pelvic anatomy in obstructive dystocia in bitches. Based on the history of previous whelpings, 20 Boston terrier and 14 Scottish terrier bitches were divided into two equal groups: normally whelping bitches and bitches with obstructive dystocia. Additional whelpings during the period of study were closely observed and the pups were immediately weighed and measured. The bitches were clinically examined and the pelvis was radiographed in ventrodorsal and lateral projections. Measurements from the radiographs showed a significantly smaller pelvic size in the bitches with obstructive dystocia compared to the normally whelping bitches. Fetal-pelvic disproportion in the Scottish terrier was mainly due to a dorsoventrally flattened pelvic canal, whereas in the Boston terrier it arose from the combination of a dorsoventrally flattened pelvic canal and big fetuses with large heads. These results suggest that radiographic pelvimetry could be used to predict a disposition for dystocia in individual bitches, and as a basis for selection of breeding animals.

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry.

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.