Milk-based Extender Research Articles

Effect of different extenders on sperm viability of buck semen stored at room temperature

The use of egg yolk containing extenders in buck semen requires the washing of the semen due to the presence of egg yolk coagulating enzyme (EYCE) in the seminal plasma of the goat. Egg yolk prevents the effects of cold shock when semen is cooled. Alternatively, storage of buck semen at temperatures above 15 °C allows use of extenders without egg yolk. The aim of the study was to evaluate the effect of different extenders without egg yolk on the viability of buck semen stored at room temperature. Ejaculates were collected once from four adult bucks and split into five aliquots each and diluted with one of five extenders: Skim milk (Mi), Laiciphos ® (Lai), Beltsville Thawing Solution (BTS), and Biociphos Plus ® (Bio-5 and Bio-20). The diluted semen was stored at room temperature, except for Bio-5, which was cooled and stored at 5 °C. Sperm motility and acrosomal status were evaluated following 3, 8, 12, 24 and 28 h. Motility and acrosomal status were significantly influenced by the extenders and storage time ( P < 0.001), while only sperm motility was influenced by the interaction between extender and storage time ( P < 0.001). Progressive motility and acrosome integrity were maintained longer in milk-based extenders, e.g. Laiciphos ®, compared to Biociphos Plus ®. Storage at 5 °C in Biociphos Plus ® was better for sperm viability than storage at 20 °C. No motile spermatozoa were recorded after 3 h of storage in BTS. In conclusion, the results of the trial indicate that milk-based extenders, especially Laiciphos ®, preserved sperm motility and acrosome integrity better than Biociphos Plus ®, when buck semen was stored at 20 °C for up to 28 h.

263BINDING OF STALLION SPERM TO EQUINE AND BOVINE ZONAE PELLUCIDAE: EFFECT OF MILK, MILK PROTEINS AND GLUCOSE

The ability of sperm to bind to zonae pellucidae (ZP) has been correlated with fertilizing capacity of sperm in several species. Limited numbers of equine oocytes are available to perform such assays. Therefore, use of heterologous ZP to perform gamete binding tests with stallion sperm would be useful. We have found that addition of 10% of skim milk-based extender with glucose [EZ-Mixin®, Animal Reproduction Systems, Chino, USA;; (EZ)] to TALP significantly increased the number of stallion sperm bound to bovine ZP. Objectives of the present experiments were to determine: (1) if stallion sperm bind in similar numbers to equine and bovine ZP, and (2) the effects of skim milk, milk proteins and glucose on sperm binding to ZP. Denuded bovine (immature) and equine (mature) oocytes were stored at 5°C in salt solution (1.5M MgCl2, 40mM HEPES, 0.1% PVP). In Experiment I, 4 ejaculates from 2 stallions were centrifuged at 300g for 6min, and sperm pellets were resuspended in 1mL of TALP or EZ. Sperm were stained with Hoechst 33342, centrifuged, and resuspended to 2×106 sperm mL−1. Oocytes were placed into droplets of 45μL of TALP (7 to 10 oocytes/trt/ejac). Extended sperm (5μL) were added to oocytes, resulting in 2times105 sperm mL−1, and the mixutre was incubated for 2h at 38.5°C. Oocytes then were pipetted in TALP to remove loosely attached sperm and observed with fluorescence microscopy;; mean numbers of sperm bound to bovine and equine ZP for TALP were 29±1.9 and 36±2.6 (P&amp;gt;0.1) and for EZ, 149±5 and 152±6.3 (P&amp;gt;0.1), respectively. More sperm bound to ZP with EZ than to ZP with TALP (P&amp;lt;0.001). Experiment II used 4 ejaculates from 4 stallions. After initial centrifugation, sperm were resuspended in 1mL of each of six extenders: TALP, EZ, TALP containing 89.5mM glucose (TG), TALP containing 163.5mM glucose (THG), TALP containing 2.4mgmL−1 of skim-milk powder (TSM), and INRA 96® (IMV Technologies, L’Aigle, France) that contains 27mgmL−1 of native phosphocaseinate. Hoechst 33342-stained sperm and bovine oocytes were processed as described for Experiment I. Treatments containing milk proteins resulted in more sperm binding (P&amp;lt;0.01) than those without milk proteins (Table 1). In conclusion, use of bovine oocytes led to similar results for equine and bovine oocytes;; therefore, bovine oocytes can be used for binding assays with stallion sperm. High concentrations of glucose increased numbers of sperm bound to ZP;; however, presence of milk or milk proteins was more effective in enhancing binding of sperm to ZP. INRA96 contains relatively low glucose (67mM) and one milk protein. Therefore, we hypothesize that native phosphocaseinate may cause increased sperm binding to ZP. Table 1 Mean sperm bound per ZP±SEM (n=38–40/group)

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen.

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.

Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell ®; IMV, L’Aigle, France) free from additives of animal origin, containing two different final glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 °C or at 15 °C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA)), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no significant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell ® (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 °C, although the results were not always statistically significant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5–10%) concentrations of egg yolk and the addition of glycerol at 5 °C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell ® containing 6.4% glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk.

Lipase activity in stallion seminal plasma and the effect of lipase on stallion spermatozoa during storage at 5 °C

Previous studies have demonstrated a detrimental effect of seminal plasma on the maintenance of motility of cooled equine spermatozoa; however, the mechanism for the adverse effect of seminal plasma during cooled storage remains undetermined. In goats, a glycoprotein component of bulbourethral gland secretion contains lipase activity that is detrimental to sperm motility when stored in skim milk-based extenders. The objective of the current study was to determine the amount of lipase activity in stallion seminal plasma and to determine the effect of added lipase on spermatozoal motility during cooled semen storage. In the first experiment, seminal plasma (1.0ml) was assayed for lipase activity based upon hydrolysis of triglycerides (olive oil substrate) into free fatty acids and subsequent titration of pH change (Sigma Diagnostic Lipase Kit). Lipase activity in stallion seminal plasma was 0.36±0.02 Sigma units/ml, (mean+S.E.M.; n=16 ejaculates from six stallions). In the second experiment, equine semen (three ejaculates from each of four stallions) was divided into five treatment aliquots. In Treatment 1, semen was extended 1:3 with nonfat dried skim milk extender (NFDSM). In treatment groups 2 through 5, spermatozoa were washed by centrifugation (300×g for 15min) and resuspended in NFDSM to a final concentration of 25×106spermatozoa/ml. Porcine pancreatic lipase (pPL) was added to Treatment 3 (10 pPL units/ml), Treatment 4 (100 pPL units/ml) and Treatment 5 (100 pPL units/ml, heat inactivated at 100°C for 5min), while Treatment 2 had no pancreatic lipase added and served as the control. Samples were cooled slowly to 5°C, and stored at 5°C until evaluation. Sperm motility was evaluated at time 0, 24, 48 and 72h by computerized semen analysis, and data were analyzed via repeated measures ANOVA. The addition of 100units/ml but not 10units/ml of pPL decreased (P<0.01) total and progressive motility of stored sperm. Heat-inactivated pPL (Treatment 5) did not significantly decrease motility of spermatozoa during storage. Because the lipase activity assayed (Sigma units) and the lipase activity added to cooled semen (pPL units) were not equivalent, pPL was assayed in the Sigma Diagnostic Lipase assay. The relationship between Sigma Units (Y) and pPL units (X) appeared to be a log-linear relationship with log(Y)=−0.912+0.007X; R2=0.90. Mean lipase activity assayed in stallion seminal plasma was equivalent to approximately 64 pPL units/ml. These data suggest that endogenous lipase activity in stallion seminal plasma may be a factor in the adverse effects of seminal plasma on cooled spermatozoa in some stallions.

Detection of lipid peroxidation in equine spermatozoa based upon the lipophilic fluorescent dye C1l-BODIPY581/591.

The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescence as detected by either fluorescence microplate reader or by flow cytometry. The addition of Fe2SO4 (0 to 0.5 mM), low concentrations of sodium ascorbate, and the addition of cumene hydroperoxide increased peroxidation of C11-BODIPY581/591. The addition of high concentrations (10 or 20 mM) of sodium ascorbate or alpha-tocopherol reduced peroxidation of C11-BODIPY581/591 during short-term incubations. During storage at 5 degrees C in a skim milk-based extender, equine spermatozoa demonstrated a progressive decline in motility and a small but significant increase in lipid peroxidation based upon ratiometric analysis of C11-BODIPY581/591. The addition of Fe2SO4 increased lipid peroxidation in cooled spermatozoa in a dose-dependent fashion and decreased sperm motility. The addition of alpha-tocopherol, however, did not reduce lipid peroxidation during cooled semen storage. These data demonstrate that the lipophilic fluorescent probe C11-BODIPY581/591 is a useful measurement of lipid peroxidation in equine spermatozoa and that there is an increase in lipid peroxidation during cooled storage of equine spermatozoa that is increased in the presence of ferrous promoters.

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 °C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac ® and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 °C seemed to influence sperm viability parameters less than storage at 20 °C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 °C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.

Advances in cooled semen technology

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4–8 °C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks’ salts, supplemented with NPPC was then developed for use with cooled/stored semen. Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 °C, and anaerobic conditions when stored at 4 °C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 °C or diluted in Kenney or INRA82 extenders and then stored at 4 °C for 24 h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 °C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 °C for 24 h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E– Z Mixin extenders then stored at 4 °C for 24 h until insemination. Experiment 1 demonstrated that at 15 °C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 °C after 24 h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 °C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% ( n=52) compared to 68% ( n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 °C (54%, n=37) as at 4 °C (54%, n=35) after 24 h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 °C (59%, n=39) seems to improve fertility per cycle compared to E– Z Mixin at 4 °C (49%, n=39, P=0.25), but this result has to be confirmed. These results demonstrate that semen diluted in INRA96 extender and stored at 15 °C can be an alternative to semen diluted in milk-based extenders and stored at 4 °C for “poor cooler” stallions. Furthermore, INRA96 extender can be as efficient at 15 °C as at 4 °C, for preserving sperm motility and fertility.

Influence of centrifugation and different extenders on post-thaw sperm quality of ram semen

Using a 2-step extension methodology to freeze ram semen, 2 freezing protocols (P1 and P2) and 3 extenders were evaluated in a split-sample experiment. The freezing protocols were tested in combination with Extenders A and B (Experiment 1), and B and C (Experiment 2). Protocol 1 included centrifugation before filling the straws to reconcentrate the diluted semen to a calculated sperm concentration of 800×10 6cells/mL. Protocol 2 involved appropriate ejaculate extension to yield 800×10 6 cells/mL as in P1, albeit avoiding centrifugation. Extenders A and B were milk-based and were supplemented with 5% egg yolk and fructose. Extender B was clarified by centrifugation (twice at 3310g/20 min). Extender C was based on TRIS-citratefructose supplemented with 20% egg yolk and clarified as described for Extender B. Final glycerol concentration was 7% for all 3 extenders. Post-thaw parameters studied were subjective motility, computer assisted sperm motility analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline assay, CTC). The overall sperm concentration (×10 6/straw) differed (P<0.001) between P1 (mean ± SD, 138.1+14.8) and P2 (216.5±13.9). Despite centrifugation, P1 appeared to be less harmful for spermatozoa than P2, yielding higher percentages of subjective motility, linearity, membrane integrity and uncapacitated spermatozoa. Due to the difference in concentrations obtained between P1 and P2, the total calculated numbers of spermatozoa having desirable characteristics were higher in samples processed as P2. In Experiment 1, P1 resulted in lower calculated numbers ×10 6in the AIdose of subjective motility (87.2+5.1 vs 125.3±5.1; P<0.05), linearity (70.6±4.3 vs 79.8±4.3; NS), intact-membrane (77.4±5 vs 108.5±5.1; P<0.001), and uncapacitated (36.5±2.5 vs 46.5±2.5; P<0.05) spermatozoa, than P2. In Experiment 2, calculated sperm numbers (×10 6/straw) were lower in P1 than in P2 for subjective motility (80.8±5.4 vs 92.0±5.4; NS), linearity (63.3±5.6 vs 73.1±5.6; NS), membrane integrity (77.7±3.6 vs 101.0±3.6; P<0.001), and uncapacitated spermatozoa (28.3±3.24 vs. 4.1±3.2; P<0.01). Extender B (clarified milk extender) was consistently better than Extender A (nonclarified milk extender) for all parameters studied, but the difference was only statistically significant for linearity after 1 h of incubation at 38° C (44.0±2.4 vs 36.2±2.4; P<0.05). Extender B was also better than Extender C (TRIS-citrate-fructose) for percentage of uncapacitated (49.7±2.2 vs 34.4±2.3; P<.001), subjective motile (57.5±2.7 vs 43.8±2.7; P<0.01), and linear motile (46.5±2.8 vs 33.7±2.8; P<0.01) spermatozoa, but not for membrane integrity (51.6±1.5 vs 51.7±1.5). It was concluded that exclusion of centrifugation, as in P2, yielded higher sperm numbers with desirable characteristics per straw. Clarification of milk-based extender (B) resulted in better post-thaw sperm quality, especially compared with TRIS-based extender (C).

Estudo da viabilidade do processo de refrigeração do sêmen canino, utilizando-se diluidores à base de leite e glicina gema

Foram utilizados 27 ejaculados de um cão, adulto, SRD, de porte médio. O sêmen foi coletado por meio de massagem peniana e dividido em três amostras iguais: GRUPO I (G I)-refrigerado, sem a utilização de meio diluente; GRUPO 2 (G2): diluído com meio à base de leite desnatado; GRUPO 3 (G3)-diluído com meio à base de glicina-gema, sem glicerol. As amostras foram avaliadas logo após a coleta (MO) quanto à motilidade, ao vigor espermático e à integridade das membranas espermáticas e, logo após, incubadas à temperatura de soe. Em seguida, as amostras foram avaliadas nos momentos:24(M I), 48(M2) e 72(M3) horas de refrigeração, para motilidade e vigor; em M3 foi avaliada também a integridade das membranas espermáticas. A análise estatística dos dados obtidos mostrou equivalência entre o G2 e o G3, ambos superiores ao G I quanto aos parâmetros estudados. A técnica de refrigeração mostrou-se viável, e os diluentes utilizados foram eficazes na preservação das funções espermáticas, durante o processo de refrigeração, na espécie canina.

Sperm Viability in Ram Semen Diluted and Stored in Three Different Extenders

Semen was collected with an artificial vagina from 4 one-year-old rams, in order to study the changes in sperm motility and membrane integrity of spermatozoa split-diluted and stored at 5 degrees C during 7 days in sodium citrate, Tris, and milk-based extenders, respectively. Sperm motility was assessed subjectively and sperm membrane integrity was determined using the fluorescent probes Calcein-AM and Ethidium homodimer. Representative samples were studied using scanning electron microscopy (SEM). The average incidence of sperm motility decreased over time in all the extenders (p < 0.001). The incidence of spermatozoa showing progressive motility and intact plasma membrane was significantly higher in semen diluted with sodium citrate than in the other 2 extenders following 4 days of dilution until the end of the study. Evaluation with SEM confirmed the findings obtained with the supra vital fluorescent dyes. The results of the present study indicated that there were no differences between sodium citrate-, Tris- or milk-based extenders when ovine liquid semen was stored at 5 degrees C during a short period (2 days). However, when semen was stored for longer time, spermatozoa in the sodium citrate-based extender sustained its viability better.

Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders

The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 μL of c-SOF+NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.

Deterioration of goat spermatozoa in skimmed milk-based extenders as a result of oleic acid released by the bulbourethral lipase BUSgp60.

The aim of the present study was to elucidate the mode of action of goat bulbourethral lipase (BUSgp60 lipase) previously identified as responsible for the deterioration of goat sperm viability in skimmed milk-based extenders. Milk fractions were purified by micro- and ultrafiltration and characterized by SDS-PAGE, thin layer chromatography, triglyceride quantitative analysis and by their ability to potentiate the lipase and the sperm-deteriorating activity of the bulbourethral lipase. Components in both the phosphocaseinate and soluble whey protein fractions enhanced the lipase activity of BUSgp60 but only the phosphocaseinate fraction, which contains triglycerides, promoted deterioration of spermatozoa in the presence of bulbourethral gland secretion. These data suggest that the sperm-deteriorating effect of bulbourethral gland secretion is due to the catalysis of triglyceride hydrolysis, and that proteins increase this activity. BUSgp60 hydrolysed milk triglycerides and triolein very effectively, and its lipase activity was enhanced by several highly purified milk proteins. The major cis-unsaturated fatty acid from milk (oleic acid) but not the major saturated fatty acid (palmitic acid) exhibited dose-dependent detrimental effects on goat spermatozoa. Therefore, the catalysis of oleic acid formation from residual milk triglycerides by BUSgp60 appears responsible for the deterioration of goat spermatozoa when unwashed semen is diluted in skimmed milk-based extenders. The precise mechanism of action of oleic acid remains to be elucidated but the drawbacks of washing buck semen might be avoided by inhibiting BUSgp60 or by depriving it of substrate.

Deterioration of goat sperm viability in milk extenders is due to a bulbourethral 60-kilodalton glycoprotein with triglyceride lipase activity.

The aim of this work was to purify, identify, and characterize the component of goat bulbourethral gland secretion (BUS) responsible for goat sperm deterioration in skim milk extender. BUS extracts promote a decrease in the percentage of motile spermatozoa, deterioration in the quality of movement, breakage of acrosomes, and cellular death of goat spermatozoa diluted in skim milk. A 55- to 60-kDa monomeric glycoprotein (BUSgp60) was purified by cation-exchange, concanavalin A, and heparin-affinity chromatography and was identified as the only BUS component responsible for the deterioration of spermatozoa in milk. The BUSgp60 was shown to display triacylglycerol hydrolase activity, and partial sequences (37 amino acids in all) exhibited 50-70% homology with sequences of various types of pancreatic lipases (PLs), especially PL-related protein 2 (PL-RP2). In addition, porcine PL produced deterioration of goat sperm properties in milk as effectively as BUS and BUSgp60. These results support the preliminary identification of the goat BUSgp60 as a bulbourethral lipase belonging to the PL-RP2 family. Since seminal plasma also contains factors favorable for sperm survival, it is envisaged that, for the best preservation of goat semen, specific inhibitors of this family of lipase could be added into milk-based extenders without eliminating seminal plasma.

Comparison of Two Semen Extenders in Terms of In Vitro Development of Bovine Embryos Following IVF

In order to conform with current EC standards with regard to antibiotic cover, the Norwegian Cattle Association is currently investigating the use of Biladyl as an alternative to the milk-based extender which has been traditionally used in Norway. A study was carried out to investigate the effect of using semen frozen with either milk extender or Biladyl on the outcome of in vitro fertilization and embryo culture. Semen from 6 Norwegian Red bulls was used. There was a significant difference p < 0.05 in terms of cleavage rate between the 2 extenders for 1 bull, 78.2% vs 94.9% for milk and Biladyl extenders, respectively, and for the overall total of 71.3% vs 76.1% for milk and Biladyl extenders, respectively. There were no significant differences in terms of blastocyst yield amongst any of the bulls. In conclusion, the results suggest that Biladyl can be used as a replacement for the traditional milk-based extender without any adverse effects on blastocyst yields following in vitro fertilization.

Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa

Experiments were conducted to determine temperatures between 24 and 4°C at which stallion spermatozoa are most susceptible to cold shock damage. Semen was diluted to 25 × 10 6 spermatozoa/ml in a milk-based extender. Aliquots of extended semen were then cooled in programmable semen coolers. Semen was evaluated by computerized semen analysis initially and after 6, 12, 24, 36 and 48 hours of cooling. In Experiment 1A, semen was cooled rapidly (−0.7°C/minute) from 24°C to either 22, 20, 18 or 16°C; then it was cooled slowly (−0.05°C/minute) to a storage temperature of 4°C. In Experiment 1B, rapid cooling proceeded from 24°C to either 22, 19, 16, or 13°C, and then slow cooling occurred to 4°C. Initiating slow cooling at 22 or 20°C resulted in higher (P<0.05) total and progressive motility over the first 24 hours of cooling than initiating slow cooling at 16°C. Initiation of slow cooling at 22 or 19°C resulted in higher (P<0.05) total and progressive motility over 48 hours of cooled storage than initiation of slow cooling at 16 or 13°C. In Experiment 2A, semen was cooled rapidly from 24 to 19°C, and then cooled slowly to either 13, 10, 7 or 4°C, at which point rapid cooling was resumed to 4°C. Resuming the fast rate of cooling at 7°C resulted in higher (P<0.05) total and progressive motility at 36 and 48 hours of cooled storage than resuming fast cooling at 10 or 13°C. In Experiment 2B, slow cooling proceeded to either 10, 8, 6 or 4°C before fast cooling resumed to 4°C. There was no significant difference (P>0.05) at most storage times in total or progressive motility for spermatozoa when fast cooling was resumed at 8, 6 or 4°C. In Experiment 3, cooling units were programmed to cool rapidly from 24 to 19°C, then cool slowly from 19 to 8°C, and then resume rapid cooling to storage temperatures of either 6, 4, 2 or 0°C. Storage at 6 or 4°C resulted in higher (P<0.05) total and progressive motility over 48 hours of storage than 0 or 2°C.

Effects of linear cooling rate on motion characteristics of stallion spermatozoa

Three experiments were designed to analyze the effects of cooling rate on survival of stallion spermatozoa in a milk-based extender, at 0 to 96 hours after reaching the desired temperature. The samples were warmed to 37°C and were evaluated by computer-assisted analysis of sperm motility. In Experiment 1, rate of cooling between 37 and 20°C was evaluated. Sperm motion was not affected by cooling at plunge, −0.42 or −0.28°C/minute. However, storage of spermatozoa at 5°C after slow cooling below 20°C was superior to storage at 20°C. In Experiment 2, 3 cooling rates from 37° to 5°C were evaluated. Cooling at either −0.05 or −0.7°C/minute was superior (P<0.05) to plunging spermatozoa to 5°C. Cooling at −0.05°C/minute rather than −0.7°C/minute maximized the percentage of motile spermatozoa and their curvilinear velocity. In Experiment 3, cooling rates from 20 to 5°C were evaluated, with all samples cooled at −0.7°C/minute from 37 to 20°C. Sperm motion was similar (P>0.05) after cooling below 20°C at −0.012, −0.05 or −0.10°C/minute, and the 2 slower rates were superior (P<0.05) to cooling at −0.3°C/minute. It was concluded that stallion spermatozoa can be cooled rapidly from 37 to 20°C, but should be cooled at ≤−0.1°C/minute and preferably at −0.05°C/min from 20 to 5°C to maintain maximum sperm survival at 5°C.

Production of monozygotic goat twins by transfer of frozen-thawed demi-embryos

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl®, Andromed® and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P ≤ 0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1−/PI− (intact spermatozoa), and Mitotracker+/YO-PRO-1− (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P ≤ 0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl® and Andromed® resulted in a greater sperm quality (P ≤ 0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P ≤ 0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl® or Andromed® are used as freezing extenders.

Progress in behavior therapy with delinquents : Jerome S. Stumphauzer. Charles C. Thomas, Publisher (301–327 East Lawrence Avenue, Springfield, Illinois 63703), 1978, 390 pp., hardcover-$29.75; softcover-$22.75

The aim of this study was to assess extenders for cooling equine semen at 5°C and to be used in assisted reproductive technologies (ARTs). Four ejaculates were obtained from each of four stallions. Gel-free semen was diluted in three different extenders: (1) SMK, an opaque skim milk–based extender; (2) SMT, a skim milk (65%) and Tyrode medium (35%); and (3) BSAG, a clear extender containing 1% bovine serum albumin. Samples were packaged (10 mL; 50 × 106 sperm/mL) and stored in a cooling device at 5°C for 12 hours. Analyses were done at 0, 4, 8, and 12 hours after cooling. Semen was analyzed for sperm motility characteristics using a computer-assisted sperm analysis, for plasma membrane and acrosome integrity and mitochondrial membrane potential, using fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide [JC-1]). Morphology was evaluated with differential interference contrast microscopy and sperm chromatin integrity by the toluidine blue technique. Data were analyzed by analysis of variance and by Tukey test, with time as a repeated measure (SAS, 1998), when P < .05 was significant. In general, milk-based extenders (SMT and SMK) showed improved maintenance of semen quality compared with BSAG. Finally, the addition of skim milk to equine semen extender for cooling at 5°C for 12 hours seems to play a crucial role in sperm preservation. Although, optically clearer extenders are desired for use in ARTs, such as sperm sexing, the milk-free extender (BSAG) is less efficient for cooled-stored equine semen.