Canine Semen Research Articles

DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa

Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase–mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.

A Novel Collection Technique for the Improvement of Semen Quality

To date, the common thread in the use of semen extenders/collection techniques, whether they are being used for fresh extended semen, chilled semen, or cryopreserved semen, is that the extenders have all traditionally been added post-collection. The objective of this study was to determine if modifying the method of collection/extension of semen to include a warmed media environment would improve semen parameters by lessening cold and pH shock. Ten canine semen samples were collected with a modified artificial vagina to allow for a true split collection into to collection containers at one time. The treatment half of the sample was collected into a measured amount of warmed extension media. The control half was collected into a dry container and no attempts to maintain temperature was used. Standard semen parameters, available sperm pool, and number of inseminations were evaluated at specific time intervals and evaluations continued until samples reached zero percent motility. Data analysis was performed with SPSS using the general linear model and appropriate t-tests. There was a difference between the treatment and control groups for Motility (P<.001), Motility by Time (P<.001), Time to Zero Motility (P<.001), Time to Last Full Insemination (P<.03), Forward Progression (P<.001), Acrosome Reaction (P<.001), Acrosome Reaction by Time (P<.02) and Viability (P<.001). There was no difference in morphology between the treatment and control groups (P>.05). Modification of the semen collection/extension procedure resulted in improved semen parameters for extended time-periods post-collection. The data suggest the described collection technique can yield significantly more motile sperm by placing the sample into a physiologically favorable environment (eliminating pH and cold shock and allowing osmoregulation to begin), thus providing more available sperm for breeding.

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates

The aim of this study was to evaluate the sperm quality in chilled canine semen using different cooling rates from room temperature (23 °C) to 5 °C and subsequently cold-stored at 5 °C for up to 96 hours. In experiment 1, semen samples from five dogs were pooled, diluted in Tris-fructose-citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5 °C using different cooling rates of 2.25, 0.9, 0.45, and 0.2 (control) °C/min. In experiment 2, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 °C/min or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72, and 96 hours of storage at 5 °C. The total motility, progressive motility, and quality of movement parameters were assessed using computer-assisted analysis system, and the percentage of viable spermatozoa was determined using flow cytometry (H-42/PI//FITC-PNA). The cooling rate did not influence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively influenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 hours at 5 °C in a Tris-fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.

Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P&lt;0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm.

Cryopreservation of canine semen after cold storage in a Neopor box: effect of extender, centrifugation and storage time

The aim of this work was to assess the combined effect of sperm centrifugation, semen extender and storage time before freezing on post-thaw sperm quality and freezability on chilled stored...

Artificial insemination in dogs and cats 2. Artificial insemination in dogs

An article in the February issue of In Practice discussed the benefits of artificial insemination in dogs and explained how to collect and preserve canine semen (In Practice, February 2014,...

EFECTO DE LA ADICIÓN DE FLUIDO PROSTÁTICO AUTÓLOGO Y HETERÓLOGO SOBRE LA CALIDAD ESPERMÁTICA DEL SEMEN CANINO

The effect of adding autologous or heterologous prostatic fluid in canine sperm and incubated at 38 °C (thermoresistance) on sperm motility and spermatic vigor was evaluated.Fifteen ejaculates were collected from mature English bulldog by digital manipulation.The sperm fraction and the prostatic fractions were kept separated during the collection.Prostatic fluids were processed and stored at -18 °C. An spermiogram was done for each ejaculate and then distributed in three aliquots of 0.5 ml and added 1 ml of autologous prostatic fluid, heterologous prostatic fluid or saline solution. The samples were incubated at 38 °C for 60 minutes measuring the progressive motility and spermatic vigor at 0, 15, 30,45 and 60 minutes. The results showed a lineal decreased for both parameters during the evaluation period, and without statistical difference between treatments or times of incubation. It is concluded that the progressive motility and spermatic vigor of canine semen diluted with prostatic fluid either analogous or heterologous were similar to those on saline solution.

Artificial insemination in dogs and cats 1. Collection and preservation of canine semen

Artificial insemination (AI) is an important reproductive technology that is increasingly used in cats and dogs to facilitate management of breeding colonies and individual animals. The basic technique of transferring...

Effects of Storage Temperature and Semen Extender on Stored Canine Semen

ABSTRACTThe objective of the present study was to determine an optimum temperature and extender for short-term transport of canine ejaculated semen. There was no significant difference in the qualities of semen diluted with two kinds of extender, egg yolk Tris-citrate fructose (EYT-FC) or glucose (EYT-GC) extender, between the 2, 8 or 12 and the 4°C control groups during storage for up to 48 hr, while the 16–24°C groups showed decreased sperm motility during storage for 48 hr. However, the 2°C group showed slightly lower sperm motility and slightly higher sperm abnormality than the 4°C group. Therefore, we concluded that semen qualities can be maintained for up to 48 hr when canine semen samples are extended with EYT-FC or EYT-GC and stored at a temperature in the range of 4–12°C.

A Systematic Review of Studies Performing the Hypo‐Osmotic Swelling Test to Evaluate the Quality of Canine Spermatozoa

The hypo-osmotic swelling test (HOS test) is a simple and inexpensive test to evaluate the functional integrity of sperm cell membranes. According to the existing literature, its simple applicability has turned it into a valuable additional parameter to standard canine semen analysis. In the recent years, much research has been conducted in this field. The aim of this systematic review was to evaluate the quality of published literature in canine reproduction concerning the HOS test. Using two distinguished databases, 38 articles were detected and analysed subsequently according to various aspects, for example study design, population, semen sampling and implementation concerning the HOS test. Although there are numerous articles available, the diagnostic value of the HOS test remains ambiguous. Until now, neither a recognized test protocol nor reliable reference values have been defined. Most of the trials evaluated show serious methodological flaws and therefore do not permit drawing reliable conclusions. According to our results, approximately half of the studies (n=20) included a sample size of five or less animals. None of the studies examined the inter- or intraobserver agreement for the HOS test. Further research is warranted including appropriate statistical methods and a sufficient number of animals to establish a standardized test protocol as well as reliable reference values. Most importantly, it is required to clarify a correlation between the HOS test and the fertilizing capacity to determine the diagnostic value of the HOS test.

Effects of green tea polyphenol on the quality of canine semen after long-term storage at 5 °C

The aim of the present study was to determine the effects of green tea polyphenol on the quality of canine semen after long-term storage at 5°C. The supplementation of a Tris-egg yolk extender with polyphenol (0.5, 0.75, or 1mg/mL) increased the motility and viability of sperm preserved for four weeks at 5°C.

Cooled Storage of Canine Semen:in vitroEffects of Different Concentrations of an Antibiotic Combination on Growth of Mollicutes

The aim of this study was to examine effects of an antibiotic combination at different concentrations on growth of mycoplasma and ureaplasma during cooled storage of canine semen (n=20). Semen aliquots were diluted with Tris-citric acid-fructose-egg yolk extender containing either 1.0g/l streptomycin and 0.6g/l benzylpenicillin (control) or a combination of gentamycin, tylosin, lincomycin and spectinomycin (GTLS-1: 0.25, 0.05, 0.15 and 0.3; GTLS-2: 0.5, 0.1, 0.3 and 0.6; GTLS-3: 1.0, 0.2, 0.6 and 1.2g/l). Samples were assessed for motility and membrane integrity by computer-assisted sperm analysis immediately after dilution and at 24, 48 and 72h of cooled storage. Morphologically, normal spermatozoa were determined, and bacterial culture was performed at 24 and 72h. Mycoplasma spp. were detected in 14 of 20 ejaculates (70%) with severe growth in 12 samples. A reduction but not total elimination of mycoplasma growth occurred in all GTLS extenders with the most pronounced reduction in group GTLS-3 (control vs GTLS-1 and GTLS-2 p<0.05, control vs GTLS-3 p<0.001). Ureaplasmas were detected in four ejaculates, and growth was reduced to the same extent in GTLS and control extender. Progressive motility in all groups, total motility in groups GTLS 1-3 and percentage of membrane-intact spermatozoa in groups GTLS 2 and 3 decreased slightly (p<0.05) over time. In conclusion, dilution of canine semen with GTLS extender has no major detrimental effects on spermatozoa during cooled storage. It reduced the growth but did not totally eliminate mycoplasmas and ureaplasmas from cooled-stored dog semen.

Spectrophotometric Analysis of the Resazurin Reduction Test as a Tool for Assessing Canine Semen Quality

Abstract The resazurin reduction test (RRT) was subjected to spectrophotometric analysis to evaluate the quality of canine semen. Twenty four samples of canine semen were analysed. The absorption peaks for resazurin and resorufin were determined at 615 and 580 nm, respectively. The RRT ratio (RRTsperm-the ratio for samples containing spermatozoa, RRTplasma-the ratio for samples containing seminal plasma) was calculated by dividing the absorbance at 580 nm by the absorbance at 615 nm. Spearman’s correlation test was used to determine the significance of correlations between the analysed sperm parameters and the results of the resazurin reduction assay. The RRT ratio was highly correlated with sperm motility (r=0.68, P&lt;0.01), progressive sperm motility (r=0.61, P&lt;0.01), the subpopulation of cells with rapid velocity (r=0.72, P&lt;0.01), and the subpopulation of cells with medium velocity (r= -0.54, P&lt;0.05). A negative correlation was observed between the reducing capacity of seminal plasma vs. sperm with plasma membrane integrity (r= -0.60, P&lt;0.01) and sperm with normal morphology (r= -0.58, P&lt;0.01). The RRT test can be used as an additional tool for evaluation of the quality of canine semen.

Season related variations in the canine semen characteristics

Season related variations in the canine semen characteristics

84 EFFECT OF A STRESSOR ON CANINE SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for human and different mammalian species (Sperm-Halomax®), based on the sperm chromatin dispersion test (SCDt); however, no studies has been performed specifically on canine frozen–thawed-stressed semen but is there for cooled semen. The aim of this work was to assess the effect of a stressor (24 h in an oven at 38°C) on canine frozen–thawed semen using the SCDt to resemble what happens in the female reproductive tract. For this purpose, ejaculates were collected by digital manipulation from 4 healthy beagle dogs and the sperm-rich fraction of the ejaculates from 3 different dogs was pooled each time. All the pooled semen samples (n = 4) used presented physiological values concerning to routine semen parameters (motility, morphology, and sperm concentration). After evaluation, semen samples were centrifuged and the sperm pellet resuspended to a final concentration of 100 × 106 sperm mL–1 in 2 steps with CaniPRO Freeze (Minitub, Tiefenbach, Germany). Sperm were slowly cooled to 5°C and then loaded into 0.5-mL plastic straws. After that, straws were frozen in liquid-nitrogen vapours for 10 min and stored into a nitrogen tank. Straws were thawed in a water bath (30 s/37°C) and incubated for 24 hours at 38°C before analysis. The sperm DNA fragmentation was assessed in fresh semen and frozen–thawed-stressed samples using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain) following the manufacturer’s instructions. Slides were stained for green fluorescence staining and 500 sperm per slide were counted using fluorescence microscopy. The sperm DNA fragmentation index (%) was compared between fresh and frozen–thawed-stressed semen samples by ANOVA. Results were expressed as mean ± standard error of the mean. The results obtained showed that subjecting thawed semen to 24 h in an oven at 38°C significantly increased (P &lt; 0.05) DNA fragmentation compared with fresh semen (2.7% ± 0.2 v. 1.4 ± 0.1%). The stress factor was performed to simulate the viability of canine thawed sperm (12–24 h) when a bitch is inseminated with frozen semen. It would be interesting to perform further studies to relate sperm DNA fragmentation and fertility of frozen–thawed canine semen. In conclusion, frozen–thawed-stressed semen samples increased the sperm DNA fragmentation index measured using a SCDt. Further studies are needed to relate sperm DNA fragmentation with fertility rates or cryopreservation success.

Intravaginal Artificial Insemination in Bitches Using Frozen/Thawed Semen after Dilution in Powdered Coconut Water (ACP‐106c)

The aim of this study was to evaluate powdered coconut water extender (ACP-106c; ACP Serviços Tecnológicos Ltda, ACP Biotecnologia, Fortaleza, Ceará, Brazil) as a diluent for freezing dog semen and the fertility after vaginal insemination of semen frozen therein. Ten ejaculates were collected from five dogs, evaluated fresh, diluted in ACP-106c, 10% egg yolk and 6% glycerol, cooled and frozen. In the first phase of the study, straws with frozen semen were thawed and immediately subjected to the same analysis as the fresh semen and, in addition, to Computer-Assisted Semen Analysis (CASA). In phase 2, 10 bitches that had been subjected to natural breeding during a preceding oestrous cycle were vaginally inseminated with thawed semen that had been re-diluted in ACP-106c. After thawing, a mean of 77% sperm motility was obtained through subjective analysis and 77.3% through CASA. Following artificial insemination, a 60% pregnancy rate was observed, resulting in a 50% parturition rate and a mean litter size of 3.4 (SEM 0.6), with 47.1% males and 52.9% females. ACP-106c can be successfully used for freezing canine semen, and vaginal deposition of such semen yields similar pregnancy rates to those reported in other studies.

Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa

The aim of this study was to determine the effect of two variants of Tris-citric acid-fructose (TCF) extender containing whole hen egg yolk (TCF-HEY) and lyophilized lipoprotein fractions extracted from ostrich egg yolk (TCF-LPF(o)) on selected biological properties of cryopreserved sperm cells. Post-thaw percentage of motile sperm (MOT) was significantly higher (P < 0.05) for TCF-HEY extender (66.3 +/- 3.2%) than for TCF-LPF(o) extender (52.4 +/- 3.4%). Moreover, there was no significant difference in the percentage of sperm with progressive motility (PMOT). Both diluents effectively preserved sperm plasma membrane integrity and mitochondrial function. However, it was observed that cryopreservation impaired the functionality of antioxidant sperm enzymes. The above was manifested by reduced SOD activity, in particular in samples preserved in the TCF-HEY extender, as well as decreased GPx activity. Both diluents inhibited the rate of lipid peroxidation in sperm plasma membrane during freezing-thawing. Our results suggest that LPF(o) is a satisfactory alternative to hen egg yolk in the extender used for canine sperm cryopreservation.

Effects of Cooling Time on Membrane Integrity and Motility of Frozen‐Thawed Canine Spermatozoa using Two Different Commercial Egg Yolk–based Extenders at Two Different Cooldown Equilibration Times

Two commercially available egg yolk-based semen extenders, one marketed for human semen freezing (HEYE) and one marketed for canine semen freezing (CEYE), were used to cryopreserve semen from single ejaculates of 11 different dogs. For each extender, a 30- and a 60-min cooldown period was used prior to the addition of the extender containing glycerol and then immediately frozen in liquid nitrogen vapours. Sperm motility was measured using a computer-assisted semen analysis (CASA) system. Sperm intact membranes were measured using SYBER-14 and propidium iodide. Semen in the HEYE cooled for 60 min had a significantly greater percentage of intact membranes than the semen in the HEYE cooled for 30 min (p = 0.02). Semen in the HEYE cooled for 60 min had significantly greater total motility (p = 0.007) and progressive motility (p = 0.004) than semen cooled for 60 min in the CEYE and semen cooled for 30 min in the HEYE (total motility p = 0.02 and progressive motility p = 0.02). Semen cooled for 60 min in the CEYE did not differ significantly in total (p = 0.6) or progressive motility (p = 0.4) than semen cooled for 30 min in the CEYE. There was no difference in total (p = 0.8) or progressive motility (p = 0.8) between the semen cooled for 30 min in the HEYE and the semen cooled for 30 min in the CEYE.

Use of Fluorescent Stainings and Flow Cytometry for Canine Semen Assessment

Over the last few decades, appreciable progress has been noted in canine semen assessment techniques. The common use of accurate and sensitive diagnostic methods, such as computer-assisted sperm analysis (CASA), flow cytometry and sperm penetration tests have become routine procedures in specialized andrology laboratories. Many fluorescent probes have been applied to the assessment of specific sperm characteristics in dogs. Flow cytometry enables the observation of cell characteristics such as size, shape and function of the spermatozoon, that can be revealed by a fluorochrome or fluorescent label. The analysis of events detected on dot plots gives accurate and highly reliable information on membrane integrity, acrosomal status, mitochondrial activity, capacitation status, lipid peroxidation, apoptosis and DNA damage. Despite the development of these modern and accurate tools, it is still questionable if the ideal method of semen evaluation, allowing predicting of the fertilizing potential of semen, has been established.

Effect of extenders on sperm mitochondrial membrane, plasma membrane and sperm kinetics during liquid storage of canine semen at 5 °C

The objective of this study was to evaluate the effect of addition of egg yolk (EY) or soy lecithin (SL) based extenders on dog sperm parameters during 10days storage at 5°C. Four ejaculates of pooled semen from three Mongrel dogs were divided into three aliquots and extended to a final concentration of 200 million sperm/mL using following non-commercial extenders: egg yolk extender (EYE) group – semen+20% Tris-EYE; soy lecithin (SL) group 1 – semen+SL extender with 0.04% SL; SL group 2 – semen+SL extender 0.4% SL. The extended semen samples were stored at 5°C and were evaluated for sperm mitochondrial membrane potential (MMP), plasma membrane integrity (%PMI) and computer assisted sperm analyzer (CASA) sperm motility parameters on days 0, 1, 2, 3, 4, 7 and 10. The extender, days of storage and extender×days of storage affected the MMP, %PMI and CASA sperm motility parameters (P<0.05). The addition of 0.4% SL extender reduced the speed of deterioration of sperm parameters evaluated in this study compared to EY and 0.04% SL extenders (P<0.05). In conclusion, the addition of 0.4% SL extender enhanced sperm preservation based on the variables evaluated in the present study compared to EYE and 0.04% SL extenders and plausibly preserves sperm quality longer.