Skim Milk Extender Research Articles

Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre-sperm fluid and the first sperm-rich jets (HIGH-1), the main sperm-rich portion (HIGH-2), the jets with low sperm concentrations (LOW), and a combined whole-ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH-2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion's own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse-phase liquid chromatography (RP-HPLC), N-terminal sequencing and mass spectrometry. The area-under-the-curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS-PAGE and densitometry. No significant differences emerged between fractions in the AUC-values of the Horse Seminal Protein-1 (HSP-1) and HSP-2 peaks, or the peak containing HSP-3 and HSP-4 (HSP-3/4). Levels of HSP-1, HSP-2 and HSP-3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60-70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.

19 FACTORS AFFECTING PREGNANCY RATE AFTER ARTIFICIAL INSEMINATION WITH EQUINE SEMEN: PRODUCTION OF THE FIRST FOAL USING FROZEN SEMEN IN KOREA

Research in the area of equine artificial insemination (AI) has led to its increased application in field trials (Loomis and Graham, 2008). However, procedures for equine semen collection, cooling and freezing of semen and artificial insemination need further improvement. This study examined the effect of semen preparation and ovulation on equine AI success. Stallion semen was collected via artificial vagina, diluted with skim-milk extender, and preserved by either cooling to 4°C or cryopreservation and storage in liquid nitrogen. Mares were examined by ultrasound for follicle development with every 12 h during oestrus. During AI, semen was inserted into uterine horn adjacent to the ovulating follicle sized over 4 cm and irregular shape. In experiment 1, mares were inseminated with either cooled, cooled-transported, or frozen–thawed semen. Cooled semen was used immediately after collection and cooling. Cooled-transported semen was used 4 h after semen collection and dilution. Semen freezing procedure was followed by Moore et al. (2006). In experiment 2, we examined the effect of buffer (skim-milk extender), which was infused into the uterus at the time of AI with frozen–thawed semen. In experiment 3, we compared AI pregnancy rates for mares ovulating spontaneously v. after treatment with hCG. Pregnancy data were analysed Chi square test where P-values < 0.05 were considered significantly different. In experiment 1, the pregnancy rates after AI using cooled, cooled-transported, and frozen–thawed semen were 60 (10/6), 50 (8/4), and 37.5% (8/3), respectively, and similar among treatments. In experiment 2, the pregnancy rate of mares infused with buffer at AI was 37.5% (8/3) which was higher than that with no buffer (0%; 6/0). In experiment 3, the pregnancy rates of mares were similar between ovulated spontaneously (25%; 8/2) and ovulated with hCG (50%; 8/4). The results suggest that equine semen that has been cooled, cooled and transported or frozen can be successfully used to establish AI pregnancy. Also, the pregnancy rates after AI can be increased by infusing buffer into the uterus at AI or by inducing ovulation with hCG. These experiments resulted in the first foal produced in Korea with frozen–thawed equine semen. This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Effect of seminal plasma on uterine inflammation, contractility and pregnancy rates in mares

There is conflicting evidence over the role seminal plasma plays in sperm transport and inflammation within the uterus of mares. In in vitro studies, seminal plasma has been shown to reduce polymorphonuclear neutrophil (PMN) function, but the opposite effect on uterine inflammation has been reported in vivo. To study the effect of seminal plasma on uterine contractility, inflammation and pregnancy rates by inseminating mares with low doses of sperm free from seminal plasma (Group 1) and containing seminal plasma (Group 2). Synchronised mares were inseminated with 50 x 10(6) sperm in either skim milk extender or seminal plasma. Uterine lavage was performed 6 h after insemination to assess the inflammatory response. The contraction frequency of the uterus was measured over a 4 min period 10 mins and 6 h after insemination, using B-mode ultrasonography. Pregnancy rates were assessed 16 days after insemination. Uterine contractions were less frequent in Group 1 mares inseminated with seminal plasma and significantly more PMNs were found in the lavage fluid of those mares. Pregnancy rates were identical in both groups (62%). This study provides evidence that seminal plasma decreases uterine contractility and increases the inflammatory response of the uterus to semen. No effect of seminal plasma on pregnancy rates was demonstrated. Mares that develop persistent mating-induced endometritis may have inherently poor uterine contractility and impaired uterine clearance. The presence of seminal plasma during breeding may not be desirable in these mares. The role of seminal plasma in problem mares warrants additional study.

Efeito da centrifugação sobre a qualidade do sêmen canino

Avaliou-se o efeito da centrifugação sobre a viabilidade do sêmen canino e compararam-se três meios de diluição pré-centrifugação. Utilizaram-se 10 ejaculados completos de 10 cães que, após a avaliação inicial, foram divididos em quatro porções (grupos). Uma das amostras, não centrifugada, formou o grupo-controle; as outras foram diluídas em três diferentes meios e centrifugadas a 800 x g por 15 minutos, formando os grupos: CPSA - constituído por sêmen centrifugado em plasma seminal autólogo; CLG - sêmen centrifugado em meio à base de leite desnatado e glicose (LG); e CPer- sêmen centrifugado em gradientes de Percoll (45% e 90%). Após a centrifugação e a eliminação do sobrenadante, procedeu-se à ressuspensão de todas as porções do ejaculado em LG e à imediata avaliação quanto à motilidade, vigor, aglutinação espermática e integridade das membranas espermáticas. Todas as suspensões foram, então, incubadas a 37ºC por 30 minutos e reavaliadas. O processo de centrifugação não causou danos aos espermatozoides e a centrifugação em meio LG melhorou a viabilidade espermática.

EFFECT OF EXTENDERS ON THE CHARACTERISTIC AND FERTILITY OF COOLD BUFFALO SEMEN

The present study was conducted to investigate the effect of semen extenders onthe characteristics of cooled buffalo semen and to determine the suitable extender which will give higher conception rate. Semen was collected from five buffalo (5-6 years) bulls twice weekly using artificial vagina. Freshly collected semen samples were evaluated and ejaculates of at least 70% individual motility and 6x106 sperm /ml were pooled, divided into three parts, diluted with tris egg yolk, citrate egg yolk or skimmed milk extender and cooled slowly to 5oC. Semen samples were evaluated for individual motility, alive sperm, plasma and acrosomal membranes integrity as well as viability index. Conception rates of buffalo cows artificially inseminated with cooled buffalo semen were also estimated. The results of the present study revealed that semen extender and storage time exert a highly significant effects on the characteristics of cooled buffalo semen with the higher values recorded for tris egg yolk diluted samples. The conception rate of cooled buffalo semen was significantly (P<0.01) higher in tris (70%) diluted samples than skimmed milk (60%) and egg yolk citrate (50%) extended samples. It was concluded that the tris egg yolk extender is the most satisfactory buffering system to improve the post-cooling semen quality in buffalo and gave the best conception rate.

Effect of Antibiotics in Extender on Bacterial and Spermatozoal Quality of Cooled Buffalo (Bubalus bubalis) Bull Semen

The present study was designed to study the effect of traditional antibiotic combination (streptomycin and penicillin; SP) and relatively modern combination of antibiotics (gentamycin, tylosin, lincomycin and spectinomycin; GTLS) in extender on bacterial control and spermatozoal quality of liquid buffalo bull semen stored at 5 degrees C. Semen collected from Nili-Ravi buffalo bulls (n = 10) was diluted with skim milk extender containing either SP (streptomycin 1000 microg/ml and penicillin 1000 IU/ml), GTLS (gentamycin 500 microg/ml, tylosin 100 microg/ml, lincomycin 300 microg/ml and spectinomycin 600 microg/ml) or negative control with no antibiotics (NA). Liquid semen was stored at 5 degrees C for 5 days. Aerobic bacteria isolated from buffalo semen were Pseudomonas aeruginosa and Staphylococcus aureus. The only facultative anaerobic bacterium isolated was Klebsiella pneumoniae. In vitro antibiotic sensitivity test revealed that Ps. aeruginosa and Staph. aureus were susceptible to gentamycin. Staphylococcus aureus and K. pneumoniae were susceptible to tylosin and linco-spectinomycin. Total aerobic bacterial count was significantly lower in semen samples treated with GTLS than those of SP on third and fifth day of storage at 5 degrees C. There was no difference (p > 0.05) in sperm motility, longevity and plasma membrane integrity (PMI) in extender containing SP or GTLS combination until the third day of storage at 5 degrees C. On fifth day of storage sperm motility, longevity and PMI was significantly better in extender containing SP compared with GTLS and NA. Intact acrosomes, and sperm head, mid piece and tail abnormalities remained similar (p > 0.05) because of antibiotics up to 5 days of storage. In conclusion, GTLS is more capable than SP for bacterial control of buffalo bull semen. Moreover, GTLS and SP are equally efficient in preserving spermatozoal quality of extended buffalo bull semen for 3 days at 5 degrees C.

Comparison of ticarcillin and piperacillin in Kenney's semen extender

Ticarcillin and piperacillin were compared to determine their effect on sperm motility and bacterial growth of equine semen samples diluted in Kenney's glucose skim milk semen extender. Each ejaculate ( n = 11) was divided into three portions and glucose skim milk semen extender solution was added. The control semen extender solution contained extended semen and no antibiotic, whereas ticarcillin and piperacillin solutions contained extended semen plus 1.0 mg/mL of ticarcillin or piperacillin, respectively. An aliquot was removed (1 h after collection) to evaluate sperm motility and microbial concentration. All three solutions were stored at 4 °C and aliquots were obtained at 24 and 48 h to determine sperm motility and microbial concentration. Mean percentages of motile and progressively motile sperm did not differ significantly among control and antibiotic-containing solutions after storage. Control-extended semen samples from ejaculates of stallions ( n = 11) were contaminated with aerobic gram-positive and gram-negative bacteria. In solutions that contained either antibiotic, growth of these microbes was inhibited after 1, 24, and 48 h at 4 °C. Semen samples from stallions ( n = 5) were extended with Kenney's glucose skim milk extender containing no antibiotic, ticarcillin or piperacillin and then inoculated with approximately 5 × 10 2 CFU/mL Klebsiella pneumoniae or Pseudomonas aeruginosa; there was no significant difference between antibiotics in the inhibition of microbial growth. In conclusion, piperacillin was an appropriate alternative to ticarcillin in extenders for equine semen.

Cryopreservation of goat spermatozoa: Comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples ( n = 110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1 h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).

Stallion semen cryopreservation using different cryoprotective agents on the skim milk trehalosa extender

Cryoprotective agents (CPAs), protect the sperm during cryopreservation. There are two general classes of CPAs, at first penetrating cryoprotectants, these pass through the sperm membrane and act both intracellular and extracellularly, and the second non-penetrating cryoprotectants, these act only extracellularly. The objective of this study was to evaluate the addition of different CPAs namely glycerol (G), combination of ethylene glycol with glycerol (EG) and dimethilformamide (DMF) using skim milk trehalosa extender. The semen collected from 3 stallions using artificial vagina twice aweeks. The semen was evaluated, centrifugated and diluted in skim milk extender supplemented with 50 mM trehalose and three different CPAs with the concentration of sperm were 200x10 6 ml -1 . Extended semen was then packed into minitub 0.3 ml and equilibrated at 4 o C for 2 hours, freeze in the liquid nitrogen vapor for 10 minutes and stored in liquid nitrogen container -196 o C. After 24 hours, the semen was thawed at 37 o C for 30 second. Results of this experiment indicated that the percentage of motile and viable sperm in skim trehalosa extender using DMF and glycerol better than combination of ethylene glycol and glycerol. Key Words : Cryopreservation, Stallion Sperm, Cryoprotectant

11 FERTILITY OF FRESH AND FROZEN - THAWED EPIDIDYMAL STALLION SPERM WITH OR WITHOUT EXPOSURE TO SEMINAL PLASMA

The aim of this study was to determine if the addition of equine seminal plasma to epididymal semen enhances its fertility before or after freezing. Thirty-two mares were randomly assigned to 5 stallions; 3 stallions were kept in Pretoria, each having 7 mares, and 2 stallions were kept at Cornell, one having 6 mares and the other 5. Mares were synchronized using 10 daily IM progesterone and estradiol injections; an Ovuplant® implant (26 mg of deslorelin; Peptech Animal Health, Sydney, NSW, Australia) was inserted under the mucosa of the vaginal vestibulum once a follicle reached a diameter of 35 mm; implants were removed after ovulation. Mares were inseminated 30 h after implant insertion. Each insemination dose consisted of 200 million progressively motile sperm and was deposited into the uterine body. Following insemination, mares were examined for ovulation at 6 hourly intervals. Fourteen days after ovulation, mares were examined for pregnancy by transrectal ultrasonography and treated with PGF2α to induce the next estrus. Seminal plasma was collected from the stallions used in the trial prior to castration, frozen, and stored. In Pretoria, stallions were castrated and one epididymal tail was flushed with seminal plasma and the other with skim milk extender; in the first cycle, half of the mares were inseminated with one of the two sperm samples. In Cornell, testes of each stallion were removed 3 weeks apart, and all mares were inseminated first with one and 3 weeks later with the other semen sample. Mares were inseminated during consecutive estrous cycles using the following sperm types: fresh epididymal sperm that had been exposed to seminal plasma (G1: 4 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); fresh epididymal sperm that had never been exposed to seminal plasma (G2: 3 mares per stallion in Pretoria, 6 and 5 mares per stallion at Cornell); frozen–thawed ejaculated sperm (G3); frozen–thawed epididymal sperm that had been exposed to seminal plasma prior to freezing (G4); and frozen–thawed epididymal sperm that had never been exposed to seminal plasma (G5). The results of inseminations with fresh epididymal semen (G1–2) of 5 stallions and the preliminary results of inseminations with frozen–thawed epididymal semen (G3–5) of 2 stallions are summarized in the Table 1. Cycles where ovulation did not occur within 12 h after insemination were excluded. The pregnancy rate of mares inseminated with fresh epididymal sperm of G1 was significantly higher (chi-square test; P &amp;lt; 0.05) than that of mares of G2. The pregnancy rate of mares inseminated with frozen–thawed ejaculated semen (G3) was similar to that of mares inseminated with frozen–thawed epididymal semen of G4 and G5 (P = 0.3). Based on these preliminary results, we conclude that the fertility of fresh epididymal sperm can be enhanced by exposure to equine seminal plasma. To determine if the same holds true for frozen–thawed epididymal sperm, more inseminations must be performed. Table 1.Results of inseminations with various semen types

Effect of Seminal Plasma Fractions on Stallion Sperm Survival after Cooled Storage

This study aimed to evaluate stallion sperm survival after 24 h of cooled storage in the presence of seminal plasma (SP) derived from the sperm-rich fractions (SRF) or sperm-poor fractions(SPF) of the ejaculate, without SP, or in the presence of SP from other stallions. Ejaculates were collected from four stallions using an automated phantom, which separated the semen into five cups. Centrifuged and washed spermatozoa from cup 2 (SRF) were mixed with skim milk extender to a concentration of 100 x 10(6) sperm/ml and then 1:1 (v/v) with SP from the stallion's own or another stallions' second (SP-SRF) or last cup (SP-SPF). Skim milk extender (K) and skim milk extender supplemented with modified Tyrode's medium (KMT) were used as control treatments. After a 24-h storage period in a transport container, spermatozoa were evaluated for motion characteristics and plasma membrane integrity by calcein acetoxymethyl (AM)/propidium iodide staining. The percentage of spermatozoa with intact plasma membranes after storage was lower in SP-SRF than in SP-SPF, and the highest in K (P < 0.05). Progressive motility (PMOT) was lower for sperm stored in SP-SRF than for sperm stored in SP-SPF (P < 0.05), but there was no significant difference in total motility (TMOT). Sperm stored in KMT (P < 0.05) registered the highest TMOT and PMOT percentages. Osmolarity was significantly higher and pH lower in K than in KMT or SP. Treatment with SP-SPF from three stallions benefited the PMOT of sperm from one stallion. These preliminary findings suggest that SP from SRFs may be more harmful during storage than SP from SPFs. Removal of SP improves sperm survival in KMT extender, and exchanging SP between stallions seems to influence sperm survival.

Effect of sperm numbers and concentration on sperm transport and uterine inflammatory response in the mare

Our objective was to determine whether the concentration of cooled sperm inseminated influenced sperm transport and intensity of the uterine inflammatory reaction 2, 4 and 24 h after insemination. Experimental subjects were 189 estrous mares with a dominant follicle ≥35 mm in diameter and no bacterial growth or neutrophils detected in uterine smears. Each mare was randomly assigned to receive one of the following intrauterine treatments (volume, 20 mL): insemination with 5 × 10 6 mL −1 or 25 × 10 6 mL −1 or 50 × 10 6 mL −1 sperm diluted in 3 mL seminal plasma (SP) and 17 mL skim milk; seminal plasma or skim milk extender. Mares in a control group received no intrauterine treatment. Mares were slaughtered 2, 4 or 24 h after insemination or infusion. Oviducts were separated from the uterus, and uterus and oviducts were then flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected for further histopathological examination. The grade of uterine fibrosis and the amount of neutrophils in the stratum compactum were evaluated. A sample of each tubal flushing was examined for sperm count, and a sample of each uterine flushing was examined for PMN count. It was concluded that compounds in the insemination dose provoked a uterine inflammatory response, which was more rapid and intense as sperm concentration increased. In contrast, sperm transport through 4 h after insemination was not influenced by sperm concentration.

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.

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 °C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 °C. Total motility after 24 h of storage was lowest in semen diluted with PBS ( p < 0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly ( p < 0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly ( p < 0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender ( p < 0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.

Motility and Plasma Membrane Integrity of Spermatozoa in Fractionated Stallion Ejaculates after Storage

With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with extender (Expt 1) or frozen in liquid nitrogen (Expt 2). In Expt 1, cup 1 was pre-sperm fluid, cups 2 and 3 sperm-rich fractions, and cup 4 sperm-poor fractions. In Expt 2, cups 1 and 2 were sperm-rich fractions, and cups 3 and 4 sperm-poor fractions. One sample (WE) represented the whole ejaculate in both experiments. Motility parameters were determined with a Hamilton-Thorn Motility Analyzer, and plasma membrane integrity was assessed using carboxyfluorescein diacetate and propidium iodide staining and fluorescence microscopy. The removal of seminal plasma lowered motility values, but not plasma membrane integrity, in both experiments. No significant differences between cups were observed after cooled storage. The cups differed significantly in most post-thaw motility parameters, and the sperm-rich fraction showed higher post-thaw motility than the whole ejaculate.

20 SPERM CHARACTERIZATION OF ASTURCON PONIES AFTER COLLECTION AND EQUILIBRATION/REFRIGERATION BEFORE FREEZING

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the convention of biological diversity, and sperm quality is known to be an important criterion in the evaluation of breeding soundness. The aim of this work was to characterize the semen of a local breed of ponies 'Asturcon' (maintained free over the mountains all year around) before its incorporation into a germplasm bank. Semen was obtained from six stallions (6–17 years of age) using an artificial vagina, 3 days/week, during 12 weeks. Immediately after collection, gel-free semen was evaluated for volume, sperm concentration, and motility. Semen motility was again evaluated after equilibration/refrigeration. For evaluation of individual (IM) and progressive motility (PM) rates, semen was diluted (20 � 106 spermatozoa/mL) and analyzed with a CASA System (SCA; Microptic S.L., Barcelona, Spain). Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (100�). Semen samples were subjected to a hypo-osmotic swelling test (HOS) test to detect the presence of swollen tails in a 100 mM citrate–fructose solution. Percentages of altered acrosomes and morphological abnormalities were determined by counting 100 spermatozoa (1000�). Then, semen was diluted and centrifuged for 10 min at 600g. After the supernatant was discarded, the pellet was re-suspended in freezing medium (skim milk extender containing 2% egg yolk and 2.5% glycerol) to a final concentration of 100 � 106 spermatozoa/mL, and equilibrated/cooled (60 min) to 4�C. Statistical analysis was carried out by means of the GLM and CORR procedures and Duncan test for means (SAS Institute, Inc., Cary, NC, USA). A significant effect between males (P &amp;lt; 0.05) on semen quality, such as volume of the ejaculate, sperm concentration, and morphological abnormalities, were detected among stallions. On the other hand, positive and significant correlations were found between the sperm motility immediately after collection and after equilbration/refrigeration (r = 0.73; P &amp;lt; 0.05); moreover, sperm motilities (both fresh and refrigerated) correlated with the results of the HOS test (r = 0.56; P &amp;lt; 0.001, and r = 0.27, P &amp;lt; 0.05, respectively). These preliminary results confirm that the sperm of the Asturcon ponies breed can be collected and will survive the equilibration/refrigeration procedures. Conservation and development of local breeds is important because they represent a unique source of genes for improving health and performance traits of industrial breeds. However, complementary studies on the ability of the stallion sperm to survive freezing/thawing procedures in rates higher than 30% are needed to ensure that genetic banks are correctly created. This work was performed in collaboration with ACPRA and Dep�sito de Sementales de Santander (Spain), and supported by RZ2004-00031-C02-01.

Equine spermatozoal motility and fertility associated with the incorporation of d-(+)-mannose into semen extender

Mannose is capable of decreasing bacterial attachment to the uterine mucosa in mares. Bacteria gain entry into the mare's uterus during breeding; therefore, a practical method to deliver mannose to the uterus is to incorporate it into semen extenders. The effect of mannose on spermatozoal motility and subsequent sperm fertilizing capability is unknown. The present study evaluated progressive spermatozoal motility in semen extender formulations incorporating mannose and assessed the fertility of mares inseminated with a mannose-containing semen extender. In Experiment 1, progressive spermatozoal motility in extender mixtures containing 0 mannose (control), 25, 37 or 49 mg/mL mannose was evaluated at 20 °C or 5 °C holding temperatures for 0, 12, 24 and 48 h post-dilution. Measures were repeated three times using five stallions of proven fertility. High concentrations of mannose in the extender affected progressive motility beyond the time and temperature effects noted in the controls. Extender containing only mannose sugar (49 mg/mL) displayed an immediate depression in progressive motility compared with controls (45.5% versus 62.9%, respectively; P < 0.001). The 37 mg/mL mannose extender had a less dramatic decrease in motility ( P < 0.05) and only after storage at 5 °C for ≥12 h (48.7% versus 58.0%, respectively). Extender with 25 mg/mL mannose performed no differently than the control formulation under all conditions. In Experiment 2, two groups of mares ( n = 11 each) were inseminated with 500 × 10 6 progressively motile spermatozoa extended in a traditional skim milk (control) extender or the 37 mg/mL mannose extender preparation. A single-cycle pregnancy rate of 72% was achieved by both groups. Present data suggest that a semen extender containing up to 37 mg/mL mannose could maintain motile spermatozoa for on-farm use and 25 mg/mL mannose concentrations preserved motility during long-term cooling. Likewise, sperm extended with up to 37 mg/mL of mannose had the same fertilizing capability as sperm in traditional extender mixtures.

439 TGF-beta2 suppression by the antisense oligonucleotide AP 12009 as therapy for high-grade glioma: safety and efficacy results of phase I/II clinical studies

A major obstacle in successful cooled semen storage is the reactive oxygen species generated in the media by the sperm cells. Recently, many reports document the antioxidant, anti-carcinogenic and anti-mutagenic properties of flavonoids when used in vitro or in vivo. The objective of this study was to determine whether supplementing diluents with the flavonoids, silibinin or catechin, would aid in maintaining the viability of caprine sperm during cooling and storage at 5 °C. Semen samples were collected from seven post-pubertal bucks using electro-ejaculation and aliquots of sperm (40×106 cells, replicated 3 times), were diluted in 9% non-fat dried skim milk extender supplemented with 0 (control), 25, 50, 75, or 100 μM of either silibinin or catechin. Diluted samples were cooled and maintained at 5 °C prior to microscopic analysis (250×) using a Makler counting chamber. A minimum of 200 sperm cells per sample were counted to determine the percentage of motile sperm following 24, 48, 72 and 96 h of incubation. An ANOVA was performed on arcsine-transformed data to determine differences in sperm motility between control and treatment levels for each flavonoid. No differences in motility were detected for the silibinin-treated samples at any of the concentrations across the four time periods. Significant differences (P<0.05) in motility were detected between the 0 (control, 34%) and the 25, 50, 75 and 100 μM concentrations of catechin (57, 53, 55 and 64% motile cells, respectively) at 96 h. These results demonstrate that the flavonoid catechin, but not silibinin, aids in maintaining the motility of cooled goat sperm in a dose dependent manner.

Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri.

Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activity in vitro. The type and amount of the SP component that suppresses sperm-polymorphonuclear neutrophil (PMN) binding in vitro was determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h after artificial insemination (AI). SP reduced binding between spermatozoa and PMNs significantly (P < 0.05). Fertile spermatozoa were suspended in SP or semen extender and used to inseminate mares 12 h after the induction of uterine inflammation. The pregnancy rate was normal (77%) when spermatozoa were suspended in SP, but was dramatically reduced to only 5% when spermatozoa were suspended in extender. The proteins from SP, blood plasma (BP) and a skim-milk-based semen extender (skim milk extender, SME) were precipitated by ammonium sulfate, resuspended in PBS and dialyzed. The effect of the precipitated proteins on sperm-PMN binding was compared with fresh, untreated SP. Both fresh SP, and isolated SP proteins reduced sperm-PMN binding (P < 0.001). Conversely, proteins isolated from either BP or SME did not reduce sperm-PMN binding. The different concentrations of SP proteins used showed a dose-dependent suppression of sperm-PMN binding. Concentrations of 1 mg/ml SP protein significantly reduced sperm-PMN binding and 6 mg/ml reduced the binding to a level similar to that observed with fresh whole SP (P < 0.001). Finally, SP protein digested with proteinase K resulted in the complete loss of SP suppressive activity confirming that the effective component is a proteinaceous substance.

Effects of the flavonoids, silibinin and catechin, on the motility of extended cooled caprine sperm

A major obstacle in successful cooled semen storage is the reactive oxygen species generated in the media by the sperm cells. Recently, many reports document the antioxidant, anti-carcinogenic and anti-mutagenic properties of flavonoids when used in vitro or in vivo. The objective of this study was to determine whether supplementing diluents with the flavonoids, silibinin or catechin, would aid in maintaining the viability of caprine sperm during cooling and storage at 5 °C. Semen samples were collected from seven post-pubertal bucks using electro-ejaculation and aliquots of sperm (40×10 6 cells, replicated 3 times), were diluted in 9% non-fat dried skim milk extender supplemented with 0 (control), 25, 50, 75, or 100 μM of either silibinin or catechin. Diluted samples were cooled and maintained at 5 °C prior to microscopic analysis (250×) using a Makler counting chamber. A minimum of 200 sperm cells per sample were counted to determine the percentage of motile sperm following 24, 48, 72 and 96 h of incubation. An ANOVA was performed on arcsine-transformed data to determine differences in sperm motility between control and treatment levels for each flavonoid. No differences in motility were detected for the silibinin-treated samples at any of the concentrations across the four time periods. Significant differences ( P<0.05) in motility were detected between the 0 (control, 34%) and the 25, 50, 75 and 100 μM concentrations of catechin (57, 53, 55 and 64% motile cells, respectively) at 96 h. These results demonstrate that the flavonoid catechin, but not silibinin, aids in maintaining the motility of cooled goat sperm in a dose dependent manner.