Dimorphic Sperm Influence Semen Distribution in a Non-copulatory Sculpin Hemilepidotus Gilberti

We used an artificial semen emission test to examine the semen transporting role of parasperm (unflagellated sperm), which are produced along with eusperm (normal sperm) by an incomplete meiosis in the marine sculpin Hemilepidotus gilberti. After separation of contents of semen (eusperm, parasperm, and seminal plasma) by centrifugation, the distance traveled by semen discharged vertically from a syringe into seawater was compared among various test semen; eusperm semen (eusperm re-mixed with seminal plasma), parasperm semen (parasperm re-mixed with seminal plasma), normal semen (eusperm and parasperm re-mixed with seminal plasma), and natural semen (unmodified semen). Parasperm semen traveled more than 1.5 times as far as the eusperm semen. The lateral dispersion width of normal semen after emission was significantly narrower than that of eusperm semen. These findings indicate that parasperm can reduce the lateral dispersion and prolong the distance semen travels. The eusperm ratio in the lower portion of the emitted semen did not differ from that in the upper portion, indicating that eusperm evenly distribute within the ejaculate and reach the lower portion of semen. Since males cannot closely approach eggs that are deposited in the narrow space between the female's belly and the spawning substrate, restraint of lateral dispersion and prolongation of the distance semen traveled would increase the number of eusperm arriving at eggs, despite reduction of eusperm in the ejaculate.     

Semen collection,cryopreservation and artificial insemination in the dromedary camel

Collection of semen with a bovine artificial vagina (AV) was attempted with each of 14 camels over a period of 2 years. Semen samples were evaluated, extended and cryopreserved. Frozen thawed semen, diluted cooled semen or whole semen was used to inseminate some female camels which were induced to ovulate with hCG. Males ejaculated semen into the AV in 74.6% collection attempts. The male copulated for at least 200s in 62.9% attempts. The remaining copulations were of shorter duration. Similarly, 49.3% ejaculates were at least 3ml of semen. Libido and donation of semen improved from December onwards and reached a peak after mid January with peak performance persisting until April. It declined during May. The majority of camels had lost libido and refuse to donate semen by the end of May. Camel semen is in gel form. While 35.9% of 203 semen samples exhibited no individual sperm motility, 28.5% exhibited low to fair grade individual sperm motility and only 35.4% exhibited >50% sperm motility. Differences existed between animals (P<0.01) and months (P<0.05) of collection, while effect of copulation time was not significant. Mass motility was not observed in camel semen. Individual sperm motility develops after liquefaction of semen. Addition of caffeine but not chymotrypsin improved the individual motility. The mean live percent sperm count and normal acrosome were 73.3+/-1.0 and 92.0+/-0.5, respectively. Only 51.1% of 45 semen samples with pre-freeze motility of >50% and 25% of 16 semen samples from low pre-freeze motility group with an overall success of 44.2% of 61 semen samples were successfully preserved. Wide variation was observed in the freezability of semen from different males. Attempts to impregnate female camels with liquid semen, frozen thawed semen and whole semen after hCG induced ovulation resulted in 0/10, 1/13 and 4/10 pregnancies.     

Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 degrees C

Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the concentration of thiobarbituric acid reactive substances (TBARS) as an indicator of lipid peroxidation in native semen and after addition of extender, cooling and storage. Semen was collected from fertile Shetland stallions. In experiment 1, activity of antioxidative enzymes was determined immediately after semen collection and after 24 h storage at 5 degrees C. Enzyme activities were measured in native semen, semen diluted with semen extender, spermatozoa resuspended after centrifugation in extender and 0.9% NaCl as well as in undiluted and extender-diluted seminal plasma. In experiment 2, TBARS concentrations were analysed during storage of semen at 5 degrees C for 24 h. Semen storage for 24 h at 5 degrees C did not change activity of the examined enzymes. Antioxidative activity was significantly higher in extended than in native semen as well as in extended plasma than in undiluted plasma. In conclusion, the addition of semen extender increases the antioxidative activity in seminal plasma of stallions. Basal antioxidative activity in native semen as well as increased activity in extended semen are maintained over 24 h storage at 5 degrees C. TBARS content did not increase during semen storage. In conclusion, lipid peroxidation does not increase substantially during semen storage. The enzymatic antioxidative activity in semen apparently prevents ROS formation and is further increased by addition of semen extender.     

The equine frozen semen industry.

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

Quality and fertility of cooled-shipped stallion semen at the time of insemination

Stallion semen processing is far from standardized and differs substantially between AI centers. Suboptimal pregnancy rates in equine AI may primarily result from breeding with low quality semen not adequately processed for shipment. It was the aim of the study to evaluate quality and fertility of cooled-shipped equine semen provided for breeding of client mares by commercial semen collection centers in Europe. Cooled shipped semen (n = 201 doses) from 67 stallions and 36 different EU-approved semen collection centers was evaluated. At arrival, semen temperature was 9.8 ± 0.2 °C, mean sperm concentration of AI doses was 68 ± 3 x 10⁶/ml), mean total sperm count was 1.0 ± 0.1 x 10⁹, total motility averaged 83 ± 1% and morphological defects 45 ± 2%. A total of 86 mares were inseminated, overall per season-pregnancy rate in these mares was 67%. Sperm concentration significantly influenced semen motility and morphology at arrival of the shipped semen. Significant effects of month of the year on volume, sperm concentration and total sperm count of the insemination dose were found. The collection center significantly influenced all semen parameters evaluated. Semen doses used to inseminate mares that became pregnant had significantly higher total and progressive motility of spermatozoa and a significantly lower percentage of morphological semen defects than insemination doses used for mares failing to get pregnant. Results demonstrate that insemination with semen of better quality provides a higher chance to achieve pregnancy. Besides the use of stallions with good semen quality, appropriate semen processing is an important factor for satisfying results in artificial insemination with cooled-shipped horse semen.     

HIV-specific CD8+ lymphocytes in semen are not associated with reduced HIV shedding

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-gamma. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-gamma ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-gamma staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-gamma semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-gamma responses in semen correlated with reduced levels of HIV RNA in semen.     

Predictors of success of semen cryopreservation in chickens

Semen cryopreservation is very important for the ex situ management of genetic diversity in birds but it is rarely used. This is partly because of the highly variable success rates, and this emphasizes the need for predictors of semen freezability. This study evaluated the ability of semen quality tests to predict the success rates of semen cryopreservation in chickens and the relationships between each test. Individual variations of in vitro quality tests of semen were compared to the fertility obtained with fresh and cryopreserved semen. The in vitro semen quality tests represented viability, integrity, motility (percentage of viable and morphologically normal cells (PVN); mass motility (MMOT) and different motion parameters including percentage of motile spermatozoa (PMOT)) and biophysical tests (OSM, resistance to osmotic stress; membrane fluidity (FLUID)). Different in vitro tests were significantly correlated between each other for fresh (MMOT, PVN and FLUID, many criteria of objective motility) and cryopreserved semen (MMOT, different objective motility parameters, PVN). Fertility was significantly correlated with PVN for fresh semen and PVN and different objective motility criteria for cryopreserved semen. Membrane fluidity, followed by PVN, PMOT and MMOT, measured on fresh semen samples was positively correlated with fertility obtained with cryopreserved semen. The combination of the first three tests explained 85% of the variability of fertility observed with cryopreserved semen. In conclusion, we showed that different in vitro tests of semen quality are of predictive value for the success rate of semen cryopreservation in the chicken, the most accurate being membrane fluidity.     

Cryogenic preservation of semen from the Aleutian Canada goose (Branta canadensis leucopareia)

Aleutian Canada geese (Branta canadensis leucopareia) were inseminated with frozen-thawed semen containing 6% or 7% dimethylsulfoxide (DMSO) resulting in 32 fertile eggs and 17 goslings; with 7% DMSO, 19 of 31 eggs were fertile. Beltsville Poultry Semen Extender (BPSE), adjusted to 270 ± 30 mOs and 7.5 ± 0.4 pH, was used to dilute semen samples and the DMSO before cryopreservation. About half of the live spermatozoa in the fresh semen (92.9 ± 2.5% live cells, laboratory studies; 87.3 ± 7.3%, insemination trials) survived the freeze-thaw process (46.7 ± 7.8%, laboratory; 33.3 ± 17.8%, insemination trials). Samples of frozen-thawed semen contained a greater percentage of bent spermatozoa (27.1 ± 8.4% of live cells) than fresh semen (14.4 ± 3.0% of live cells). Fecal- and urate-contaminated semen (a common problem when collecting goose semen) reduced the sperm motility score from 3.2 ± 0.6 to 2.7 ± 0.7 and number of live spermatozoa in frozen-thawed semen from 49 ± 9% to 24 ± 18%. Other variables examined that had less of an effect on semen quality included semen extenders, semen holding temperature, dilution and equilibration, relationship between hour of semen collection and level of semen contamination, and the relationship between season and sperm concentration.     

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

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

Analysis of bovine sexed sperm for IVF from sorting to the embryo

The objective of this study was to characterize bovine semen parameters and determine the best IVF conditions to produce a maximal percentage of blastocysts. Four types of semen were analyzed with CASA and flow cytometry: fresh and frozen non-sexed semen; fresh and frozen sexed semen. Semen was obtained from four Holstein bulls and two ejaculates from each bull were analyzed. Oocytes from slaughterhouse ovaries were matured and fertilized in vitro with all types of semen (for sexed semen, 2, 5 or 10 μg/mL heparin was added to the IVF media while for non-sexed semen, 10 μg/mL was added in the IVF medium). Presumptive zygotes were co-cultured with Buffalo rat liver cells in Menezo's B2 medium, and cleavage rates at Day 2, and blastocyst rates at Day 7 of culture, were recorded. Sexed semen resulted in fewer blastocysts than non-sexed semen (P < 0.05), and certain bulls performed better in IVF. Freezing, and not sexing, had a more significant negative effect on semen quality. Compromised semen quality due to sexing and/or freezing can explain the reduced in vitro blastocyst rates when using frozen-thawed sexed semen. Sexed semen that appeared more capacitated seemed to require less heparin in IVF than sexed semen that appeared less capacitated to produce a maximal percentage of blastocyst. Flow cytometry sorting eliminates spermatozoa that possess compromised DNA, and therefore the reduced fertility seen in vitro is not due to an increased percentage of spermatozoa with compromised DNA. This study describes tools that can monitor semen parameters to optimize IVF conditions and thus obtain maximal blastocyst rates.     

In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars

The objective of this study was to compare the in vitro fertilizing capacity of porcine spermatozoa from fresh and frozen-thawed semen and frozen-thawed epididymal spermatozoa obtained from identical boars. Prior to IVF, fresh spermatozoa were capacitated in TCM 199. Frozen semen samples were stored in 0.25-ml plastic straws using a lactose/glycerol/orvus-es-paste extender. Cumulus-oocyte-complexes (COC) obtained from superovulated prepuberal gilts were fertilized in vitro within 2 h after aspiration with one of the semen samples. After final dilution for IVF, frozen-thawed epididymal semen samples showed motility rates (72.2 +/- 5.6%) similar to those of spermatozoa in fresh semen (76.4 +/- 4.5%), while sperm motility decreased in frozen-thawed ejaculated semen (40.2 +/- 9.4%). Considerable individual differences in sperm motility between boars were observed for ejaculated semen but not for epididymal semen. Enhanced fertilizing capacity of frozen-thawed epididymal spermatozoa was confirmed by pronucleus formation and cleavage rates, with significantly more embryos developing to the 2- and 4-cell stages compared with the groups fertilized with fresh or with frozen-thawed ejaculated semen (59.7 vs 14.6 and 16%). In conclusion, consistent in vitro fertilization rates with minimal semen variability are obtained using frozen-thawed epididymal semen. Following a modified freezing protocol, epididymal spermatozoa can easily be frozen in small containers for IVF, with higher resultant motility and fertilization rates than with ejaculated semen.     

Human sperm cryopreservation in cancer patients: Links with deprivation and mortality

Evidence is mounting for a relationship between human semen quality and environmental/lifestyle/socioeconomic factors including long term health outcomes such as mortality. The relationship between pre-freeze and post-thaw semen quality in cancer patients and these factors are unknown. Frozen semen from 217 cancer patients was thawed and analysed using a validated CASA method. Post-thaw quality was matched and compared with WHO semen analysis performed prior to storage. The English Indices of Deprivation 2010 were matched with patients and then examined for relationships with pre-freeze and post-thaw semen quality. There is a relationship between semen quality and deprivation in cancer patients. Compared with pre-freeze semen quality, post-thaw semen quality has a stronger relationship with deprivation. Sperm cryopreservation may have potential as a systemic health diagnostic test and is predictive of cancer patient mortality.     

Quantitative assessment of the probability of bluetongue virus transmission by bovine semen and effectiveness of preventive measures

Given that bluetongue (BT) may potentially be transmitted by semen, that the disease has significantly expanded in recent years, and that millions of doses of cattle semen are annually traded throughout the world, the transmission of bluetongue virus (BTV) by semen could have severe consequences in the cattle industry. The hypothesis that infected bulls could excrete BTV in their semen led to restrictions on international trade of ruminant semen and the establishment of measures to prevent BTV transmission by semen. However, neither the risk of BTV transmission by semen nor the effectiveness of these measures was estimated quantitatively. The objective of the study was to assess, in case of introduction of BTV into a bovine semen collection centre (SCC), both the risk of BTV transmission by bovine semen and the risk reduction achieved by some of the preventive measures, by means of a stochastic risk assessment model. The model was applied to different scenarios, depending on for example the type of diagnostic test and the interval between the controls (testing) of donor bulls, or the rate of BTV spread within the SCC.Enzyme-linked immunosorbant assay (ELISA) controls of donor bulls every 60 days seemed to be an ineffective method for reducing the risk of BTV transmission in contrast to polymerase chain reaction (PCR) tests every 28 days. An increase in the rate of spread within the SCC resulted in a reduced risk of BTV transmission by semen. The storage of semen for 30 days prior to dispatch seemed to be an efficient way of reducing the risk of transmission by semen.The sensitivity analysis identified the probability of BTV shedding in semen as a crucial parameter in the probability of BTV transmission by semen. However, there is a great degree of uncertainty associated with this parameter, with significant differences depending on the BTV serotype.     

Microscopic and flow cytometric semen assessment of Dutch AI-bucks: effect of semen processing procedures and their correlation to fertility

This study was done to determine the effects of processing techniques on the quality of semen from Dutch AI-bucks with the view on improving pregnancy rates after artificial insemination (AI) with liquid or frozen-thawed semen. Motility of spermatozoa was estimated under a microscope whereas the percentage live spermatozoa and the percentage live spermatozoa with intact acrosomes were determined by means of flow cytometry. Aspects of semen processing that were investigated are storage temperature of liquid semen (i), the effect of glycerol on liquid-stored semen (ii), removal of seminal plasma (iii) and type of extender (iv). The correlation between semen quality and fertility rates in inseminated does was also investigated. The percentage motile spermatozoa in semen stored in liquid form for 72 h progressively declined over time, irrespective of whether storage occurred at 4 or 18 degrees C. The percentage motile spermatozoa in semen stored at 18 degrees C was similar to that in semen stored at 4 degrees C if stored for 24 h but lower if stored for 48 h. Goats differ in the sensitivity of their spermatozoa to the deleterious effects of glycerol. Neither the removal of seminal plasma nor the type of extender had any effect on semen quality before freezing but semen frozen in a Tris-citric acid-glucose (TCG) buffer with egg yolk without removal of the seminal plasma had better quality after thawing than semen frozen in another diluent or after removal of seminal plasma. Remarkably no significant correlation between fertility and membrane integrity of spermatozoa could be found. Thus, although integrity assays for spermatozoa are useful to asses resistance to semen handling, the validity of these assays for predicting fertility is questioned.     

Can external quality control improve pig AI efficiency?

External quality control programmes carried out by central laboratories have been long established in human andrology with the aim of enhancing the accuracy and reproducibility of semen assessment. Compared to human, demands on boar semen assessment in AI stations are more complex, with the need both to identify boars with poor ejaculate quality and to monitor individual boar differences for semen storage. Additionally, appropriate assessment serves as a control instrument to ensure the security and efficiency of semen processing. Despite current limitations regarding the ability of sperm assays to estimate the potential fertility of males, it is evident that boar fertility is related to certain conventional semen tests, e.g. sperm morphology. In central studies carried out on stored semen from 11 AI stations, flow cytometric assessment of plasma and acrosome membrane integrity proved to be more sensitive in detecting sperm damage associated with ageing and temperature stress as compared to light microscopy. Membrane integrity of stored semen differed between AI stations indicating significant influences of semen processing on sperm quality. Thus external control of semen quality in reference laboratories may be useful to monitor the efficiency of internal semen quality control in individual AI stations, to identify males with lower semen quality and/or poor response to semen storage, and to verify the precision of sperm counting. The possibility that central laboratories with sufficient resources may be able to identify functionally different responding sperm subpopulations for better estimation of fertility is discussed. Ideally, external quality control schemes for AI stations would comprise application of validated tests with high relevance for fertility (including bacterial status), analysis of semen processing on the AI station, and training courses for laboratory personnel.     

Successful pregnancies with directional freezing of large volume buck semen

Artificial insemination with frozen-thawed buck semen shows variable results which depend on many factors related to semen quality and the cryopreservation processing. We conducted experiments based on a new freezing method, directional freezing, of large volumes (8 ml). In the first experiment semen from three Saanen bucks, ages 1-2-years-old and genetically selected for milk improvement, was frozen individually. Two to three-years-old Saanen females (n = 164) were synchronized with controlled internal drug release (CIDR), pregnant mare serum gonadotrophin (PMSG) and prostaglandin. Double cervical inseminations were performed with frozen-thawed semen and fresh semen as control. In the second experiment we used pooled, washed frozen semen to examine the effect of washed seminal plasma. The motility after washing was 80-90% and after thawing was 55-65% for all bucks. The sperm concentration increased with the collections and the advance into the breeding season from 1.9 x 10(9) to 4.4 x 10(9) cell/ml average. Two inseminations were carried out at 8h intervals. The first insemination was performed at 32 h after CIDR withdrawal with fresh and frozen-thawed semen. Pregnancy rates were assessed by ultrasonography conducted 40 and 90 days post-insemination (from three bucks). Results were 58, 67, 50% with fresh semen, and for frozen semen were 33, 37 and 53%; these results were significantly different in one of the three bucks (P < 0.005). In the second experiment with pooled, washed semen the pregnancy rate was 41.6%, which compared with the average results of the frozen semen in the first experiment 38.9% no significant difference was found. We conclude that freezing buck semen in large volumes (8 ml) is possible. Cryobanking of buck semen will facilitate a genetic breeding program in goats and preservation of biodiversity. Washed semen did not improve the fertility of the semen when Andromed bull extender is used.     

The value of canine semen evaluation for practitioners

Compared to human medicine, little is known in canine medicine regarding specific findings on semen evaluation and their correlation with fertility. Suggestions to optimize quality of semen evaluation in veterinary practice include creating standardized protocols for evaluation of all semen parameters and updating those protocols as needed; creating some form of quality control for the clinic laboratory; educating owners about our inability to predict with 100% accuracy whether dogs with poor semen quality never could impregnate a bitch or whether dogs with excellent semen quality always could impregnate a bitch; and generating protocols for further diagnostic work-up for those dogs with abnormal semen quality.     

Evaluating the success of sex-sorted semen in US dairy herds from on farm records

These data summarize on-farm records of dairy herds (n = 211) using sexed semen. Sexed semen was predominantly used at first and second service in virgin heifers, which is reflected in younger ages at AI and at calving. Conception rates at first service averaged 47% for Holstein heifers and 53% for Jersey heifers, which were ∼80% of that achieved with conventional semen. Analysis of inter-estrus intervals provides no evidence that cycle lengths are extended by use of sexed semen. Among singleton births, 89% were reported as female offspring and this rises to 90% for gestation lengths within a normal 265-295 d range. Age at calving appeared to interact with calf sex and semen type to influence the incidence of stillbirths. Semen type had no effect on the incidence of stillbirths among heifers delivering female calves. However, the incidence of stillbirths among heifers delivering male calves was greater for those conceived from sexed semen and was only partially explained by age at calving. Because the incidence of male calves from sexed semen is only 10%, the total incidence of stillbirths was not affected by semen type. In conclusion, failure to differentiate sexed from conventional semen in data recording and preferential bias in use of sexed semen in younger, more fertile females makes legitimate comparisons of sexed and conventional semen in the commercial setting difficult. When used in Holstein heifers, the average first service conception rate achieved with sex-sorted semen was 47%, which appeared to ∼80% of that achieved with conventional semen in the same herds. The percentage of female calves (89%) was consistent with expectations. After adjusting for age at calving, sexed semen had no affect on the total incidence of stillbirths, however the source for an apparent increased incidence of stillbirth among male calves born from X-sorted sperm populations requires further investigation.     

A polymerase chain reaction for the detection of Brucella canis in semen of naturally infected dogs

The objective was to evaluate a PCR assay for the detection of Brucella canis in canine semen, comparing its performance with that of bacterial isolation, serological tests and PCR assay of blood. Fifty-two male dogs were examined clinically to detect reproductive abnormalities and their serum was tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR assays were performed on blood and semen samples. The findings of the semen PCR were compared (Kappa coefficient and McNemar test) to those of blood PCR, culture of blood and semen, RSAT, and 2ME-RSAT. Nucleic acid extracts from semen collected from dogs not infected with B. canis were spiked with decreasing amounts of B. canis RM6/66 DNA and the resulting samples subjected to PCR. In addition, semen samples of non-infected dogs were spiked with decreasing amounts of B. canis CFU and the resulting suspensions were used for DNA extraction and amplification. Of the 52 dogs that were examined, the following tests were positive: RSAT, 16 (30.7%); 2ME-RSAT, 5 (9.6%); blood culture, 14 (26.9%); semen culture, 11 (21.1%); blood PCR, 18 (34.6%); semen PCR, 18 (34.6%). The PCR assay detected as few as 3.8 fg of B. canis DNA experimentally diluted in 444.9 ng of canine DNA (extracted from semen samples of non-infected dogs). In addition, the PCR assay amplified B. canis genetic sequences from semen samples containing as little as 1.0 x 10(0) cfu/mL. We concluded that PCR assay of semen was a good candidate as a confirmatory test for the diagnosis of brucellosis in dogs; its diagnostic performance was similar to blood culture or blood PCR. Furthermore, the PCR assay of semen was more sensitive than the 2ME-RSAT or semen culture. Examination of semen by PCR should be included for diagnosis of brucellosis prior to natural mating or AI; in that regard, some dogs that were negative on serological and microbiological examinations as well as blood PCR were positive on PCR of semen.