Skim Milk-glucose Research Articles

Development of a New Amycolatopsis orientalis Strain for High-Yield Eremomycin Production

A multistep random UV mutagenesis of an eremomycin-producing strain Amycolatopsis orientalis VKPM Ac-1125 with rather low productivity (0.5 g/L) and the further selection of the most high-yield mutant strains have resulted in a highly productive E 13-26 strain. After optimization of the fermentation medium by multifactor analysis, the most efficient composition for the fermentation medium was established, %: glycerol, 8; soybean meals, 2.0; MgCl2, 0.02; KH2PO4, 0.02; KNO3, 0.6; CaCl2, 0.3; skim milk powder, 0.46; and glucose, 0.8. The maximum productivity of this strain on the optimized medium reached 3.42 ± 0.17 g/L. Therefore, the obtained strain is considered to be very promising for further use in the selection and development of technology for the large-scale eremomycin production.

Effect of month of conception on fertility of mares inseminated with jackass semen

Fertility obtained by cross-breeding mares (Equus caballus) with jackasses (Equus asinus) was evaluated. Two extenders, containing skim milk-glucose or egg yolk-glycine were used to study the fertility of mares inseminated with diluted jackass semen (T1 and T2) or diluted and cooled semen at 5°C for 12 hours (T3 and T4). A total of 272 cycles of 208 mares of undefined breeds were evaluated, being uniformly distributed between groups. The cycles were controlled by transrectal palpation and teasing, and mares were inseminated every Tuesday, Thursday and Saturday (three times/week), from the detection of a follicle with 3.0 to 3.5cm diameter in one of the ovaries until ovulation. Pregnancy was detected using transrectal palpation, teasing and ultrasound exams every 14 days. The extenders had no effect on fertility (P>0.05). Pregnancy rates for the first cycle were 64.52%, 61.11%, 50.72% and 54.17% and pregnancy rates/cycle were 63.64%, 54.55%, 52.69% and 47.06%, respectively, for T1, T2, T3 and T4. Differences in pregnancy loss rates between groups and effect of month of conception on fertility were found. Pregnancy loss rates were significantly higher (P<0.05) in January (38.46%) and in February and March (52.38%), with an average of 33.09%. The results indicate that mares conceiving at the end of the physiological reproduction time, carrying a mule embryo, are more susceptible to pregnancy loss.

Development of an artificial insemination protocol in llamas using cooled semen

The objective of this study was to design an AI protocol using cooled semen to obtain pregnancies in the llama. Each raw ejaculate was subdivided into four aliquots which were extended 1:1 with: (1) 11% lactose–egg yolk (L–EY), (2) Tris–citrate-fructose–egg yolk (T–F–EY), (3) PBS-llama serum (S-PBS) and (4) skim milk-glucose (K). Each sample reached 5°C in 2.5h and remained at that temperature during 24h. Percentages of the semen variables (motility, live spermatozoa) in ejaculates and samples cooled with L–EY were significantly greater than those obtained when cooling with the other extenders; therefore this extender was used (1:1) for all inseminations. Females were randomly divided into four groups (A–D) according to insemination protocol. Group A: females were inseminated with a fixed dose of 12×106 live spermatozoa kept at 37°C. Group B: females were inseminated with a fixed dose of 12×106 live spermatozoa, cooled to 5°C and kept for 24h. Group C: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C and kept for 24h. These groups (A–C) were inseminated between 22 and 24h after induction of ovulation. Group D: females were inseminated with the whole ejaculate (variable doses), cooled to 5°C, kept for 24h and AI was carried out within 2h after ovulation. Pregnancy rates were 75%, 0%, 0% and 23% for groups A, B, C and D respectively. These results indicate that it is possible to obtain llama pregnancies with AI using cooled semen and that the success of the technique would depend on the proximity to ovulation.

Efeito do número de inseminações artificiais sobre a fertilidade de éguas inseminadas com sêmen asinino diluído e resfriado a 5ºC por 12 horas de armazenamento

Estudou-se o efeito do número de inseminações sobre a fertilidade de éguas inseminadas com sêmen asinino diluído, resfriado e armazenado. Os ciclos foram acompanhados por palpação retal e rufiação, sendo as inseminações realizadas às terças, quintas e sábados (três vezes por semana), a partir da detecção de um folículo de 3,0 a 3,5cm de diâmetro, em um dos ovários, até a ovulação. O sêmen de cinco jumentos da raça Pêga foi diluído nos diluidores de leite em pó desnatado-glicose ou glicina-gema de ovo, resfriado a 5ºC e armazenado por 12 horas, sendo a dose inseminante de aproximadamente 400x10(6) espermatozoides móveis (no momento da diluição final, pré-resfriamento). De acordo com o número de inseminações artificiais (IA)/ciclo, os resultados de 195 ciclos, referentes a 140 éguas, foram agrupados em 1IA, 2IA e 3 ou mais IA. As taxas de concepção, ao primeiro ciclo, foram de 50,0%, 62,1% e de 41,0% e, após quatro ciclos, de 47,7%, 55,6% e de 42,9% para 1IA, 2IA e 3 ou mais IA, respectivamente (P>0,05). O número de IA/ciclo, utilizando sêmen asinino resfriado, não teve efeito sobre a fertilidade das éguas.

Efeitos do local de deposição do sêmen e do intervalo inseminação/ ovulação sobre a fertilidade de éguas inseminadas com sêmen fresco diluído

Estudou-se o efeito do intervalo da inseminação artificial à ovulação sobre a fertilidade de éguas, considerando principalmente o local de deposição do sêmen. Os intervalos foram de 48 horas e 24 horas antes da ovulação e de 48 horas e 72 horas entre as duas últimas inseminações. As inseminações foram realizadas no ápice do corno uterino, por via intravaginal profunda, com 1/5 da dose inseminante utilizada para o corpo do útero (15 mL-GI). As éguas foram rufiadas diariamente e inseminadas às segundas, quartas e sextas-feiras, a partir de um folículo de 3,0 a 3,5 cm de diâmetro, com sêmen fresco diluído em diluidor de leite desnatado-glicose. Não houve diferença nas taxas de concepção/ciclo entre as inseminações realizadas no corpo (42,86%) e no ápice (45,95%) com concentrações médias de 489 e 102 milhões de espermatozoides móveis. Não foram observadas diferenças entre os grupos experimentais no mesmo intervalo inseminação artificial/ovulação, no entanto, quando as taxas de concepção foram comparadas entre intervalos em mesmo local de deposição do sêmen, taxas de concepção inferiores estiveram relacionadas às inseminações realizadas antes da ovulação. Por outro lado, as inseminações realizadas antes e após a ovulação, independentemente dos intervalos inseminação/ovulação, resultaram em melhores taxas de concepção. As concentrações espermáticas/dose inseminante podem ser significativamente reduzidas, sem prejuízo à fertilidade, quando o sêmen é depositado próximo à papila tubárica. Entretanto, há necessidade de inseminações antes e depois da ovulação para que se obtenham melhores taxas de fertilidade quando se utiliza sêmen de baixa viabilidade no trato genital da égua, visando ao estabelecimento de um reservatório espermático adequado no momento da ovulação.

Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10 6 sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700 g for 15 minutes (700 g) or 600 g for 12 minutes (600 g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400 g for 7 minutes (400 g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher ( P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400 g versus 600 g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700 g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400 g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher ( P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400 g compared with those centrifuged in 50-mL tubes at both 600 g and 700 g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.

Freezing equine semen: the effect of combinations of semen extenders and glycerol on post‐thaw motility

We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility. A randomised 2 x 3 block design was used. Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen. There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% (P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF (P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol (P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%. In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.

A Comparison of the Ability of Three Commercially Available Diluents to Maintain the Motility of Cold Stored Stallion Semen

The objective of this study was to compare the ability of three commercially available extenders to promote poststorage motility of stallion spermatozoa stored at 5°C with and without centrifugation to remove the seminal plasma. Diluents tested included skim milk glucose (SKMG), INRA 96, and VMD-Z. All diluents were tested with (-SP) and without (+SP) centrifugation to remove most of the seminal plasma. In experiment I, after 48 and 72 hours of storage, total (TM) and progressive (PM) motility values were higher ( P ≤.05) for those aliquots subjected to the INRA 96-SP as compared with either SKMG treatment. After 72 hours of storage, PM of spermatozoa stored in VMD-Z-SP was superior to that of spermatozoa stored in SKMG regardless of the presence of seminal plasma ( P ≤.05). In the second experiment, after 48 hours of storage, PM of spermatozoa subjected to the INRA 96-SP and VMD-Z-SP treatments were superior ( P ≤.05) to those for all treatments that had been stored without removal of seminal plasma. Removal of the seminal plasma and resuspension of the sperm pellet with either INRA 96 or VMD-Z resulted in TM after 48 hours of storage that were similar to those obtained after 24 hours of storage.

Fertilidade de éguas inseminadas no corpo ou no ápice do corno uterino com diferentes concentrações espermáticas

Foram utilizados 72 ciclos estrais de 37 éguas mestiças, com idade variando de quatro a 20 anos, distribuídas ao acaso em dois grupos experimentais (G I e G II) para estudar o efeito do local de deposição do sêmen no sistema genital sobre a fertilidade. As inseminações no ápice do corno uterino (G II) foram realizadas por via intravaginal profunda, utilizando-se a pipeta de inseminação IVI pippette (75cm), contendo 1/5 (3mL) da dose inseminante utilizada para o corpo do útero (15mL-G I). As éguas foram rufiadas diariamente e inseminadas às segundas, quartas e sextas-feiras, a partir de um folículo de 3,0 a 3,5cm de diâmetro, com sêmen fresco diluído em diluidor de leite desnatado-glicose. Apenas um garanhão de 20 anos e de fertilidade conhecida foi utilizado. As taxas de concepção/ciclo para as inseminações realizadas no corpo (42,9%-15/35) e ápice do corno uterino (45,9%-17/37) com concentrações médias de 489 e 102 milhões de espermatozoides móveis, respectivamente, não foram diferentes (P&gt;0,05).

Artificial Insemination with Canine Spermatozoa Frozen in a Skim Milk/Glucose-Based Extender

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.

Effect of pyruvate and lactate on motility of cold stored stallion spermatozoa challenged by hydrogen peroxide

Numerous workers have shown that motility of cold stored spermatozoa from some stallions can be improved by removing most of the seminal plasma by centrifugation and resuspending the spermatozoa in an extender consisting of skim milk glucose extender (SKMG) supplemented with a salt media such as Tyrode's or phosphate-buffered saline. The salt media must contain pyruvate and lactate. In an effort to test the hypothesis that pyruvate may be acting as an antioxidant, a series of experiments were conducted using a H 2O 2 challenge to artificially produce damage due to lipid peroxidation. Results of these experiments indicated that addition of lactate or pyruvate and lactate to SKMG-Tyrode's media was not able to prevent the detrimental affects of H 2O 2. The addition of lactate to the SKMG-Tyrode's media resulted in an improvement of post-storage motility; however, increasing the concentration of pyruvate did not further improve motility. Therefore, because lactate dehydrogenase has been shown to be correlated with motility, and lactate has been shown to be preferred as an energy source by spermatozoa from other species, the beneficial effects of lactate and pyruvate as components of a modified SKMG extender are probable as energy sources.

Effect of Quercetin on Capacitation Status and Lipid Peroxidation of Stallion Spermatozoa

Lower fertility of preserved stallion semen may be caused by damage to the spermatozoa or premature capacitation during storage. We investigated the use of the flavonoid, quercetin, with a standard skim milk extender for storage of stallion spermatozoa to prevent premature capacitation and acrosome reaction and to reduce the damage to spermatozoa from lipid peroxidation. Several concentrations of quercetin (0.05, 0.1, 0.2, 0.3 mM) were mixed with skim milk-glucose extender, and the antioxidant effectiveness was assessed using xanthine–xanthine oxidase challenge for 21 h. The effect of quercetin on capacitation status of sperm was assessed during a 4 h incubation at 33°C, and a storage trial at 5°C for 6 days. In addition, the ability of sperm stored in quercetin extender to undergo capacitation and acrosome reaction was assessed using heparin and A23187. Lipid peroxidation in the sperm after challenge was inhibited by any concentration of quercetin in the medium, while 0.1 and 0.3 mM quercetin were most effective at preventing capacitation and acrosome reaction during a 33°C incubation. During storage at 5°C for 6 d, quercetin addition to the extender significantly reduced the proportion of sperm capacitation. Heparin had no effect on capacitation status in either extender, but A23187 increased the proportion of sperm stored in the quercetin extender undergoing capacitation and acrosome reaction. In conclusion, quercetin in the standard skim milk–glucose extender for equine semen during storage reduces lipid peroxidation of the sperm, maintains internal adenosine triphosphate (ATP) concentration, and prevents premature capacitation of sperm cells before insemination while still allowing the sperm to capacitate and acrosome react after insemination.

105 ALTERING SEMEN EXTENDER AND GLYCEROL CONCENTRATION TO OPTIMIZE RESULTS AFTER CRYOPRESERVATION OF EQUINE SPERMATOZOA

After cryopreservation, spermatozoa from many stallions may have a lower capacity to fertilize an oocyte than fresh or cooled semen. The aim of this study was to evaluate a standard panel of semen extenders and varied concentrations of the cryoprotective agent (glycerol) to optimize sperm survival rates after cryopreservation. Semen was collected from Quarter Horse stallions (n = 3) from March to May 2006 (6 collections per stallion). Semen was filtered immediately after collection, and sample volume and sperm concentration were measured. A drop of raw semen was placed on two prewarmed slides to estimate the percentage of progressively motile sperm. The semen sample was then diluted to 100 � 106 spermatozoa/mL with a dried skim milk glucose extender (EZ Mixin Original Formula; ARS, Chino, CA, USA) or a chemically defined, milk-free diluent (INRA 96; IMV, Maple Grove, MN, USA). After 1 h of slow cooling and equilibration to 4�C, semen samples were centrifuged for 10 min at 400g. A defined volume of supernatant was removed, so that a concentration of 1000 � 106 spermatozoa/mL was obtained after resuspension of the sperm pellet. A 150-�L aliquot of semen was then added to specified quantities of the same semen extender used after semen collection and cryopreservation medium (Cryoguard�; Minitube, Verona, WI, USA) to obtain final glycerol concentrations of 2, 3, and 4%. This also gave a concentration of 100 � 106 spermatozoa/mL. After equilibration for 1 h at 4�C, spermatozoa were loaded into 0.5-mL straws and frozen in liquid nitrogen vapor. After 10 min, straws were plunged into liquid nitrogen. Semen was thawed at 37�C for 30 s and evaluated as prior to cryopreservation. Mean total semen volumes were 56, 11, and 60 mL in the 3 stallions. Their respective mean sperm concentrations were 124 � 106, 505 � 106, and 161 � 106 sperm/mL, respectively. Mean percentages of progressively motile sperm prior to cryopreservation were 64, 89, and 72%, respectively. With the paired Student&amp;apos;s t-test, percentages of progressively motile sperm after cryopreservation were evaluated with respect to semen extender and concentration of glycerol used. Mean overall progressive motility of spermatozoa after cryopreservation differed significantly between the two extenders and was 46% for INRA 96 and 35% for EZ Mixin OF (P &amp;lt; 0.001). Using EZ Mixin OF as semen extender, the best mean post-thaw progressive motility was achieved with 4% glycerol (39%) and differed significantly from that with 2% glycerol (32%; P &amp;lt; 0.01). When INRA 96 was used (49% for 4%, and 42% for 2% glycerol), there was no difference. This results provide evidence that, during freezing of equine spermatozoa, there is a significant effect of the semen extender and the concentration of the cryoprotectant on post-thaw sperm motility. We therefore suggest mini-freezing trials prior to freezing large numbers of sperm to find the semen extender and glycerol concentration that provides optimal survival rates.

Studies on motility and fertility of cooled stallion spermatozoa.

This study on extended, cooled stallion spermatozoa aimed to compare the ability of three extenders to maintain sperm motility during 24 h of preservation, and to describe pregnancy and foaling rates after artificial insemination (AI) of stallion spermatozoa stored and transported in the extender chosen from the in vitro study. After 6 and 24 h of preservation, motility, both subjective and evaluated by the motility analyzer (total, progressive and rapid), was lower in non-fat, dried skim milk-glucose than in both other extenders: dried skim milk-glucose added to 2% centrifuged egg yolk, and ultra high temperature treated skim milk-sugar-saline solution added to 2% centrifuged egg yolk (INRA82-Y). Rapid spermatozoa and sperm velocity parameters, after 24 h, were significantly higher in INRA82-Y. In the fertility trial, semen collected from three Maremmano stallions, diluted in INRA82-Y, and transported in a refrigerated Styrofoam box, was used to inseminate 56 mares of the same breed. Pregnancy rates after the first cycle and per breeding season were significantly higher for the 31 mares inseminated in three AI centres (54.8 and 80.6%, respectively) than for the 25 mares inseminated at the breeder's facilities (28.0 and 52.0%). Foaling rates were not significantly different between the AI centres mares (54.8%) and the other mares (44.0%). In conclusion, INRA82-Y yielded satisfactory pregnancy and foaling rates, especially when employed in the more controlled situation of an AI centre, and can therefore be included among those available for cooled stallion semen preservation.

Advances in cooled semen technologies: seminal plasma and semen extender

This study evaluated motility and fertility of uncentrifuged and centrifuged equine semen following dilution in a skim milk-glucose extender with or without supplemental Tyrode’s medium. In addition, the effect of seminal plasma addition to each extender was evaluated. For Experiment 1, motility of 48 h cooled, stored spermatozoa was evaluated following eight dilution treatments: uncentrifuged and diluted 1:4 (v/v) in skim milk-glucose extender (EZ Mixin CSTJ; CST-1:4) or in CST supplemented 65:35 (v/v) with modified Tyrode’s medium (KMT-1:4); uncentrifuged and diluted to 25×10 6 spermatozoa/ml in CST (CST-1:9) or in KMT (KMT-1:9); centrifuged and diluted in CST with 0% seminal plasma (CST-0) or 20% seminal plasma (CST-20) or centrifuged and diluted in KMT containing 0% seminal plasma (KMT-0) or in KMT containing 20% seminal plasma (KMT-20). Sperm motility parameters evaluated included percentage of total motile sperm (% TMOT), percentage of progressively motile sperm (% PMOT), curvilinear velocity (VCL) and straight-line velocity (VSL). Mean % PMOT was lower ( P<0.05) for spermatozoa extended in CST-1:4 compared to CST-1:9, whereas, all motility parameters were reduced ( P<0.05) in KMT-1:4 compared to KMT-1:9. Spermatozoa extended in CST-1:4 had greater % TMOT, % PMOT and VSL ( P<0.05) than in KMT-1:4. Spermatozoa extended in CST-1:9 had greater ( P<0.05) % PMOT than in KMT-1:9, however, VCL was greater ( P<0.05) in KMT-1:9. Mean VCL and VSL were lower ( P<0.05) for spermatozoa extended in CST-0 compared with CST-20, whereas, spermatozoa extended in KMT-0 had greater ( P<0.05) % TMOT, % PMOT and VSL compared to spermatozoa extended in KMT-20. Mean % TMOT and % PMOT were greater ( P<0.05) in CST-20 compared to KMT-20, however, KMT-0 increased ( P<0.05) velocity measures (VCL and VSL) compared to CST-0. In Experiment 2, fertility of centrifuged spermatozoa diluted in either CST-20 or KMT-0 was similar ( P>0.05). We conclude that modified Tyrode’s medium was not detrimental to establishment of pregnancy. Use of modified Tyrode’s medium may improve spermatozoal motility and pregnancy rates for cooled transport of semen from stallions in which all seminal plasma must be removed because of suspected toxic effects of seminal plasma on spermatozoal viability, however, Tyrode’s medium may be detrimental to sperm motility when seminal plasma is present.

Efeito do número de inseminações artificiais sobre a fertilidade de éguas inseminadas com sêmen diluído, resfriado a 14ºC e transportado

Estudou-se o efeito do número de inseminações sobre a fertilidade de éguas inseminadas, três vezes/semana (segundas, quartas e sextas-feiras), com sêmen diluído, resfriado e transportado, de apenas um garanhão. As éguas foram inseminadas a partir da detecção, pela palpação retal, de um folículo de 3,0 a 3,5 cm de diâmetro, em um dos ovários, até a ovulação. Utilizaram-se para o transporte o sêmen diluído no diluidor leite desnatado-glicose e o conteiner "Celle" modificado, sendo a dose inseminante de 400 x 10(6) espermatozóides móveis, no momento da diluição final, pré-resfriamento. De acordo com o número de inseminações artificiais (IA) utilizadas/ciclo, os resultados de 148 ciclos, de 100 éguas, foram agrupados em: 1 IA, 2IA, 3 IA e 4 ou mais IA. A eficiência de prenhez foi de 4,29; 5,04; 5,67; e 3,43, para 1, 2, 3 e 4 ou mais IA, respectivamente. As características foliculares diferiram em relação à freqüência de inseminações com os maiores valores observados no grupo de éguas inseminadas quatro ou mais vezes. Concluiu-se que menor velocidade de crescimento folicular e maior diâmetro do folículo ovulatório estiveram associados a maior número de inseminações artificiais/ciclo. Na presença de uma concentração espermática adequada, o número de inseminações/ciclo não exerceu influência sobre a fertilidade.

Effect of different processing techniques on motility and acrosomal integrity of cold-stored stallion spermatozoa

Summary Two experiments were conducted to test whether stallionand/or semen processing techniques influenced spermatozoal motility and acrosomal status following cold storage. Ejaculates from each of 18 stallions (N=54) were collected and split. In Experiment I, a skim milk-glucose extender (SKMG) was added to the semen following a 5, 15 or 30 minute delay post-collection. Following each delay, sperm were packaged at a final concentration of 25 million progressively motile sperm per ml (PMS/ml) in a commercially available skim milk-glucose extender (SKMG). In Experiment II, sperm were packaged at concentrations of 25, 50, and 75 million PMS/ml both in the presence and absence of seminal plasma (SP) utilizing SKMG and SKMG plus PBS, respectively. In both experiments, aliquots were cooled, stored, and the percentage of progressively motile and acrosome intact spermatozoa were determined at 24 and 48 hours post-collection. In Experiment 1, delayed dilution resulted in a lower recovery of PMS. In Experiment II, removal of SP resulted in higher percentages of PMS following cold storage. Increasing the concentration of spermatozoa during packaging decreased the percentage of PMS; however, removal of SP reduced the harmful effects on spermatozoa motility. These data suggest that reducing the time that spermatozoa remain in an undiluted state and removal of SP maximize recovery of progressively motile, acrosome-intact spermatozoa. In addition, individualizing the processing techniques for each stallion may enhance spermatozoal survival following cold storage.

Comparison of the longevity of motility of stallion spermatozoa incubated at 38°C in different capacitating media and containers

This study was designed to compare the effects of different media and containers on longevity of motility of spermatozoa during in vitro incubation at 38°C in either air or 5% CO 2 atmosphere. Three ejaculates were collected from each of 4 stallions. The media tested were skim milk-glucose, modified Krebs/Ringer and Hank's salts solution for incubation in an air atmosphere, and modified Krebs/Ringer and Brackett and Oliphant (BO) defined medium for incubation in a 5% CO 2 atmosphere. All samples were incubated in 5-mL borosilicate glass tubes filled with 3 mL of extended spermatozoa, 5-mL borosilicate tubes filled with 6 mL (topped) of extended spermatozoa, 35-mm Petri dishes filled with 3 mL of extended spermatozoa, and 35-mm Petri dishes with 200-μL microdroplets of extended spermatozoa under sterile mineral oil. For all treatments, individual samples were removed at 2, 4, 6 and 12 h of incubation to determine the percentage of motile cells. Overall, spermatozoa incubated in Petri dishes in both 3-mL and microdroplet treatments had significantly higher motility than those incubated in glass tubes (P<0.01). At 6 and 12 h of incubation in Petri dishes, progressive motility was significantly higher for spermatozoa extended in the Hank's salts solution than in the other media. Both the medium and container used significantly affected the longevity of motility of spermatozoa incubated at 38°C.

Assessment of fresh extended equine semen samples using spectrophotometry and resazurin

The purpose of this study was to determine if spectrophotometric assessment of resazurin dye in fresh extended equine semen samples was associated with spermatozoal parameters. This technique could be beneficial to veterinarians and horse producers for evaluating semen samples prior to artificially inseminating a mare. The reducible dye resazurin (blue color) is reduced via an oxidation-reduction reaction in the presence of metabolically active spermatozoa to resorufin (pink color), and upon further reduction to dihydroresorufin (colorless). Sixty semen samples were collected from six stallions (5 Quarter Horse and 1 Arabian) using a Missouri style artificial vagina. Sample aliquots were diluted using a 1:30 (semen: extender) ratio with a non-fat dry skim milk (NFDSM) glucose extender T. The diluted sample was then assessed microscopically at 250x to determine concentration, the number of motile, and progressively motile spermatozoa/mL. The remainder of the sample was diluted at a 1:1 (semen: extender) ratio prior to dye incubation and spectrophotometric analysis. The resazurin dye (50 μL from a 0.338 mM solution) was added to 4 (2 mL) aliquots of extended sample, thoroughly mixed, and incubated at 37°C. Butyl alcohol (4.8 mL) was added at five-minute increments (0,5, 10, and 15 minutes) to stop spermatozoal metabolism and draw the color out of the sample. Each aliquot was then vortexed prior to centrifugation at 700xg to extract the butanol color layer. Spectrophotometric absorbance values (615 nm) of the butanol color layer were recorded. Relationships between spectrophotometric absorbance values and spermatozoal parameters were assessed using correlation analyses on square root transformed data. At the 0 minute incubation time there were no associations between spermatozoal parameters and spectrophotometric absorbance values. However, at the five minute incubation time the spectrophotometric absorbance values were negatively correlated with concentration (r=−0.31; P=0.02), number of motile (r=−0.27; P=0.04) and progressively motile (r=−0.30; P=0.02) spermatozoa/mL. At the 10 minute incubation time negative correlations were observed between the spectrophotometric absorbance values and concentration (r=−0.48; P=0.0001), number of motile (r=−0.45; P=0.0004) and progressively motile (r=−0.46; P=0.0002) spermatozoa/mL. At the 15 minute incubation time negative correlations were also found between spectrophotometric absorbance values and concentration (r=−0.52; P=0.0001), number of motile (r=−0.50; P=0.0001) and progressively motile (r=−0.52; P=0.0001) spermatozoa/mL. Spectrophotometric absorbance values were associated with spermatozoal parameters at the 5, 10, and 15 minute incubation times.

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 × 10 9 spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 × 10 9 spermatozoa) or with 1 × 10 9 spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 × 10 9 progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 °C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 × 10 9 progressively motile spermatozoa that had been cooled to 5 °C and stored for 24 h. Group 3 mares were inseminated once with 2 × 10 9 progressively motile spermatozoa that had been cooled to 5 °C and stored for 24 h. All mares were given 2500 IU iv hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered im 10 mg of PGF 2α and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 × 10 9 progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 × 10 6 motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 °C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 × 10 9 progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 × 10 9 progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 × 10 9 progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 × 10 progressively motile spermatozoa given on two consecutive days. However, a shorter interval (≤24 h rather than >0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.