Tris Extender Research Articles

Insemination with Sex Sorted Fresh Bovine Spermatozoa Processed in the Presence of Antioxidative Substances

Flow cytometrically sex sorted spermatozoa are reduced in their fertilizing capacity, particularly when stored either in cooling extender or after freezing in liquid nitrogen. So far, preservation methods for sorted spermatozoa have differed only marginally from procedures used for unsorted semen. In the present study, a TRIS extender was modified to balance major cell damage caused by the sorting process and by liquid storage of the sorted spermatozoa. The new extender, containing a combination of antioxidants (AO) and bovine serum albumin (BSA), significantly increased the lifespan and fertilizing capacity of sex sorted spermatozoa. No significant differences were observed between unsorted controls and sorted samples for motility and status of sperm membranes as tested by fluorescein-isothiocyanat-peanut agglutinin/propidium iodide (FITC-PNA/PI). Acrosome integrity of spermatozoa was significantly better when semen was stored at 15 degrees C for 24 and 48 h in an extender containing AO with or without BSA as compared with controls (p < 0.05). There were no significant differences, in pregnancy rates of heifers inseminated at a natural oestrus, between unsorted controls (16/24, 66.7%) and both sorted groups (AO + BSA: 18/31, 58.1% and AO-BSA: 12/22, 54.5%). Additionally, it was shown for the first time that artificial insemination (AI) with liquid sexed bull spermatozoa stored for 72 h after sorting can result in pregnancy rates similar to AI with non-sorted semen.

266 BIRTH OF CANINE OFFSPRING FOLLOWING INSEMINATION OF A BITCH WITH FLOW-SORTED SPERMATOZOA

Different traits and temperaments that are sex-related often underlie the selection of guide dogs used for auditory and/or visually impaired humans. The objective of this project was to select X-chromosome-bearing canine sperm by means of flow cytometry/cell sorting based on DNA content (X chromosome- bearing canine sperm contain 3.7% more DNA than Y-chromosome-bearing sperm) and use the sorted sperm for artificial insemination (AI) to produce live offspring. This technology has proven reliable for sex selecting offspring using AI in a number of species and is of great commercial value to food animal livestock production. To test the efficacy of sorted canine sperm, a 2-year-old nulliparous Labrador retriever bitch in observed heat was monitored for plasma progesterone levels for a baseline rise to 2.8 ng mL–1, indicative of the LH surge. Progesterone levels more than doubled (5.8 ng mL–1) 2 days later, suggesting ovulation. One ejaculate was collected from a proven Labrador retriever stud on each of Days 2, 5, and 6 following the estimated LH peak. Sperm (200 million) were diluted in TRIS buffer without egg yolk (pH 7.4) and stained with 97.4 µm Hoechst 33342 in 1-mL aliquots for 45 min in a 34.5�C water bath and then diluted to 100 million sperm mL–1 with 4% egg yolk TRIS (pH 6.2) containing 0.002% food coloring dye (FD&amp;C #40). Sperm were sex-sorted using an SX MoFlo™ sperm sorter (Dako, Fort Collins, CO, USA), operated at 40 psi and using a TRIS-based medium (pH = 6.8, 370 mOsm kg–1 water) as sheath fluid: 10 000–20 000 events s–1 and sort rates of 800–2000 sperm s–1. Sorted sperm were concentrated by centrifugation for 20 min at 850g and, after the supernatant was removed, were extended with 20% egg yolk TRIS extender (pH 6.8) to a total volume of 2 mL. Transcervical insemination of fresh, sorted sperm occurred within 9 h of the semen collection. An additional 1 mL of 20% egg yolk TRIS extender was used to flush the insemination catheter. The respective number of X-chromosome-bearing sperm inseminated per day was 46.2, 20.3, and 18.2 million, totaling 84.7 million sperm, and the weighted purity mean across the 3 sort days was 82%. Pregnancy was ascertained by ultrasound 30 days post-AI. Five puppies (3 female and 2 male) were delivered via natural birth 62 days after first AI. Only 60% of the offspring were of the predicted sex due to an 18% probability of chance, given the weighted mean purity of the sorted sperm and for a litter of 5 puppies. This demonstrates that fresh canine sperm can be separated by sex on the basis of DNA content, and transcervical insemination of low numbers of non-frozen sorted canine sperm can be used to produce live offspring. This work was funded by XY, Inc.

Comparação entre a água de coco em pó (ACP®) e o Tris como diluidores na criopreservação do sêmen de cães

O presente trabalho comparou a água de coco em pó (ACP®) com o diluidor Tris na criopreservação do sêmen canino através da avaliação clássica e do teste de termorresistência. Cinco reprodutores caninos foram submetidos a duas coletas de sêmen através de manipulação digital. As frações espermáticas foram avaliadas quanto à sua coloração, volume, concentração, motilidade, vigor e morfologia espermática. As amostras de sêmen foram divididas em duas alíquotas: a primeira foi diluída em Tris e a segunda em ACP®. Ambos os diluidores continham 20% de gema de ovo. As amostras foram refrigeradas, adicionadas de glicerol (6%), envasadas em palhetas de 0,25mL, criopreservadas em vapores de nitrogênio, e finalmente armazenadas em nitrogênio líquido. Após uma semana, foi realizada a descongelação a 38ºC por 1min em banho-maria, sendo realizadas novas avaliações de motilidade, vigor e morfologia espermática. As amostras foram mantidas a 38ºC no banho-maria e avaliadas aos 15 e 30 min após descongelação. Não foram observadas diferenças significativas entre os dois diluidores no decorrer dos procedimentos de criopreservação e descongelação, bem como durante o teste de termorresistência, em relação às características avaliadas. O Tris e o ACP® foram eficientes em conservar 50% de espermatozóides móveis e 70% de espermatozóides morfologicamente normais após a descongelação. Assim, o ACP® pode ser utilizado como diluidor alternativo para a criopreservação do sêmen canino.

Effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status

The aim of this study was to evaluate the effect of post-thaw dilution with autologous prostatic fluid on motility parameters, longevity and acrosome status of frozen–thawed dog spermatozoa. After semen collection, seminal plasma was separated by centrifugation and stored frozen until use. Sperm pellets were diluted in two steps with an egg yolk–Tris extender to a final concentration of 5% glycerol and 0.5% Equex STM Paste. After thawing, semen was diluted 1:2 either with Tris buffer or with the autologous prostatic fluid. Motility was evaluated using a phase contrast microscope and a computer-assisted motility analyser system immediately after thawing and at hourly intervals up for 4 h at 38 °C. The status of acrosomes was assessed with Spermac ® stain at thawing and after 2 h of incubation. Motility and straight line velocity were initially higher in prostatic fluid-diluted samples (0 h and 0 and 1 h, respectively), but decreased to values similar to those of Tris-diluted samples in a time-dependent manner. In contrast, both the curvilinear velocity and amplitude of lateral head displacement were lower in prostatic fluid-diluted samples (1 and 3 h and 0, 1 and 3 h, respectively). The dilution did not have any significant effect on the percentage of acrosome-intact spermatozoa at either thawing or after 2 h. The pattern of motility of prostatic fluid-diluted samples suggests a reduction in hyperactivated motility with time, even though prostatic fluid neither prolonged spermatozoa longevity nor had any effect on the status of spermatozoa acrosomes.

Comparing ethylene glycol with glycerol for cryopreservation of canine semen in egg-yolk TRIS extenders

The objective of this work was to evaluate the possibility of substituting glycerol (G) for ethylene glycol (EG) when cryopreserving dog semen. A total of 15 ejaculates from 13 dogs was pooled into five samples and frozen in egg-yolk Tris extenders with variable ethylene glycol and glycerol concentrations, with or without Equex ® STM Paste. Two widely used glycerol extenders (Uppsala Equex II and Norwegian) were utilized as controls. Semen quality parameters assessed after thawing were total subjective motility (TSM), computer assisted sperm analysis (CASA), eosin-nigrosin staining, and flow cytometry (FC) after staining with the PI/Fitc-PSA (fluorescein isotiocianate conjugated with the agglutinin of Pisum sativum, PSA) fluorochromes. No advantages were seen in using EG to replace G when freezing dog semen or combining EG and G in the freezing medium. The Uppsala Equex II provided the best overall post-thaw parameters, followed by the egg-yolk Tris experimental extender with 5% EG and Equex ® STM Paste. The extender with 4% EG produced similar results to the Norwegian extender. High correlations ( r > 0.98) were obtained between eosin-nigrosin staining and FC, as well as between subjective and computerized motility assessment ( r > 0.90).

In vitro fertilization of in vitro matured canine oocytes using frozen–thawed dog semen

Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates ( P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).

Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides

The aim of this study was evaluate the survival ability of canine and feline spermatozoa maintained within epididymides stored at 4 °C for 24, 48 or 72 h in sterile isotonic saline solution (SAL) or a Tris–egg yolk (TEY) storage medium. Fifteen domestic dogs and 15 cats were neutered and their testes were placed in TEY or SAL and stored at 4 °C for either 24, 48 or 72 h. Sperm samples were obtained by cutting the cauda epididymides into a Tris extender and were evaluated for motility, velocity, viability, plasma membrane integrity, and acrosome morphology. In dogs, there were no significant differences between storage media for motility, plasma membrane integrity, viability and velocity. However, dog sperm stored in TEY had better acrosome morphology compared to sperm stored in SAL ( P < 0.05). Dog sperm recovered at 72 h had a reduction in all parameters studied compared to those recovered at 24 h ( P < 0.05). In cats, sperm recovered from epididymides stored in TEY had higher motility, plasma membrane integrity and velocity at all times compared to those stored in SAL ( P < 0.05). Cat sperm recovered at 72 h had reduced motility, acrosome morphology, viability and velocity compared to those recovered at 24 h ( P < 0.05). The addition of TEY to canine epididymal sperm, thus, had a better protective effect than SAL only on acrosome morphology. In cats, in contrast, TEY had a better protective effect than SAL on all epididymal sperm parameters studied. In both species, sperm recovered at 72 h had a significant reduction in all parameters studied compared to those recovered at 24 h.

Pengaruh Penambahan Trehalosa dalam Pengencer Tris terhadap Kualitas Semen Cair Domba Garut (Ovis aries)

This study was aimed to examine the quality of chilled-cement of Garut ram which was diluted with Tris extender and various trehalose concentrations. Cement was collected from three mature rams using an artificial vagina once a week. After initial evaluation, the cement was divided into four parts and diluted with Tris extender only (control), Tris extender + 0.20% (TR0.2), Tris extender + 0.40% (TR0.4), and Tris extender + 0.60% (TR0.6) trehalosa respectively. The extended-cement was stored at 5 o C. Parameters of chilled-cement quality examined were percentage of sperm motility, live sperm, and intact plasma membrane (IPM), which were evaluated every day for four days. The results showed that addition of trehalose did not significantly improved the percentage of sperm motility, live sperm, and IPM (P>0.05) of Garut ram sperm stored in refrigerator at 5 o C for 4 days. In conclusion, the addition of trehalose does not enhance the quality of chilled-cement of Garut ram.

Role of various sugars in improving frozen semen quality of Garut ram

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thawed process. The present study was conducted to examine the effects of addition of various sugars in Tris extender on sperm cryosurvival of Garut ram. Semen was collected using an artificial vagina from three mature rams once a week. Immediately after initial evaluation, semen was divided into five parts and diluted with Tris extender (control), Tris extender + 0.4% dextrose, Tris extender + 0.4% raffinose, Tris extender + 0.4% trehalose, and Tris extender + 0.4% sucrose, respectively. Semen was loaded in to 0.25 ml mini straw with the concentration of 200 million or 800 million motile spermatozoa per ml. Semen was equilibrated at 5 o C for three hours, then frozen and stored in liquid nitrogen for seven days. Quality of processed-semen including percentages of motile spermatozoa (MS), live spermatozoa (LS), intact acrosome cap (IAC), and intact plasma membrane (IPM) were evaluated after dilution, equilibration, and thawing, respectively. Data were analyzed using completely randomized design with five treatments and six replicates. Means were compared significant difference test at 0.05 significant level. Results of this research showed that there was no significantly difference (P>0.05) between treatments for all sperm quality parameters after dilution and equilibration. Mean percentages of post thawing MS, LS, IAC, and IPM for dextrose (54.00; 68.00; 66.60, and 57.83%), raffinose (50.00; 64.33; 61.80, and 61.75%), trehalose (50.83; 65.67; 61.40 and 57.75%), and sucrose (49.00; 66.80; 58.50 and 58.50%) were significantly (P<0.05) higher than control (40.83; 52.67; 54.60, and 49.40%) respectively. In conclusion, addition of 0.4% dextrose, raffinose, trehalose or sucrose in Tris extender are effective in improving frozen semen quality of Garut ram. Key Words: Sugars, Tris extender, Frozen Semen Quality, Garut Ram

Role of seminal plasma in maintaining quality of ram epididymal spermatozoa preserved at low temperature (3–5oC)

Cauda epididymal spermatozoa could be used as an alternative source of gamete in the application of various reproductive technologies, because the spermatozoa is motile and has ability of fertilizing the oocyte. The purpose of this research was to examine the effect of addition of seminal plasma on the quality of ram cauda epididymal spermatozoa preserved at 3–5 o C. Collected-spermatozoa was divided into five parts then centrifuged at 3,000 rpm for 30 min and the supernatant was removed. One-fifth was diluted with Tris extender (Tris) and the remaining was added 0.5 ml ram seminal plasma then diluted with Tris extender (PS-Tris), 15% AndroMed + 85% distilled water (PS-AM15), 20% AndroMed + 80% distilled water (PS-AM20), and 25% AndroMed + 75% distilled water (PS-AM25), respectively. Quality of collected-spermatozoa including spermatozoa concentration (SC), percentages of motile spermatozoa (MS), live spermatozoa (LS), abnormal spermatozoa (AS), cytoplasmic droplet (CD), and intact plasma membrane (IPM) were evaluated. Percentages of MS, LS, and IPM of diluted-spermatozoa was evaluated every day during preserved at 3–5 o C for three days. Results of this study showed that mean SC, MS, LS, AS, CD, and IPM of fresh spermatozoa were 11,660 million/ml, 65, 81, 7.6, 10.2, and 82.2%, respectively. Addition of seminal plasma in cauda epididymal spermatozoa prior to dilution could maintain the quality of spermatozoa during preserved at 3–5 o C for three days. At day-4 of storage, percentages of MS, LS, and IPM for PS-Tris (43, 58, and 59.2%), PS-AM20 (40.5, 53.75, and 53.75%), and PS-AM25 (40, 54.8, and 55.2%) were significantly (P<0.05) higher than Tris (21, 34.8, and 33.6%) and PS-AM15 (20, 40, and 42.2%). In conclusion, addition of seminal plasma in ram cauda epididymal spermatozoa prior to dilution with Tris or 20 and 25% AndroMed extenders could maintain the quality of spermatozoa during preserved at 3–5 o C for three days, and could be used for artificial insemination or in vitro embryo production programs. Key Words: Cauda Epididymal Spermatozoa, Seminal Plasma, Tris, AndroMed, Ram

118 CRYOPRESERVATION OF CONVENTIONAL AND SEX-SORTED BULL SPERM USING A DIRECTIONAL FREEZING METHOD

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P &amp;lt; 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P &amp;lt; 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P &amp;lt; 0.01) and progressive (16.3% vs. 4.3%; P &amp;lt; 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P &amp;lt; 0.05), viability (60.5% vs. 40.8%; P &amp;lt; 0.01), and acrosomal integrity (86.9% vs. 71.0%; P &amp;lt; 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit.

14 MOTILITY AND FERTILITY OF BULL SPERMATOZOA FROZEN IN EGG YOLK EXTENDER SUPPLEMENTED WITH LACTOFERRIN

Lactoferrin (Lf) is secreted by the prostate, seminal vesicles, and epididymis, and appears to represent a major component of the sperm-coating antigens that protect sperm. The aim of this study was to determine the effects of Lf used in semen extender on motility and fertility of bovine frozen semen. Semen samples were collected from three fertile Japanese Black sires. The semen collected was cooled and extended with egg yolk–Tris extender supplemented with glycerol and Lf (from bovine milk) to give final concentrations of 7% and 0, 10, 100, 500, 1000, or 2000 �g mL-1, respectively. After being extended, the semen was packaged in 0.5-mL straws, frozen, and transferred into liquid nitrogen. At least one week later, the frozen semen was thawed and washed mTALP containing caffeine. Motile sperm were separated by discontinuous 2-step (40 and 80%) Percoll gradient centrifugation. Sperm motility was calculated as the percentage of motile sperm separated by Percoll gradients relative to pre-separated sperm. Sperm motility character (motile sperm, progressive sperm) was analyzed by a CASA system (Hamilton Thorne Biosciences, Beverly, MA, USA). In the following experiment, we examined the effect of Lf added in the extender on improvement of semen fertility. The frozen semen from one sire extended with or without 500 �g mL-1 of Lf was prepared (99 straws with Lf, 99 straws without), distributed to three inseminators, and used to inseminate to 198 cows. The sperm motility and motility character were analyzed by ANOVA and Fisher&amp;apos;s exact test. The field trials were evaluated by chi-square test. After selection of motile sperm with the Percoll gradient, examination of sperm motility revealed that addition of Lf in the extender increased sperm motility significantly (P &amp;lt; 0.05) compared to that in the control without Lf: 51.2–53.8% in semen with added Lf (100–2000 �g mL-1) vs. 39.6% in the control. CASA analysis showed that motile sperm and progressive sperm were improved significantly by the addition of Lf; the peak level was 500 �g mL-1 of Lf in the extender. The percentage of motile sperm in semen with added Lf was higher significantly (P &amp;lt; 0.01) than that of the control: with Lf of 100 and 500 �g mL-1, 50.8 and 56.0%, respectively, vs. 39.7% without Lf. The percentage of progressive sperm was also significantly higher (P &amp;lt; 0.05) than that of the control: with Lf of 100 and 500 �g mL-1, 18.8 and 19.0%, respectively, vs. 13.5% without Lf. In the next experiment, we performed field trials to assess the availability of Lf for artificial insemination. In total, the group with Lf showed some but not a significant increase in fertility rate (delivery cow/inseminated cow) compared with the control group without Lf: 56.6% vs. 52.5%, respectively. In conclusion, these results suggest that addition of Lf in sperm extender may be useful for improvement of sperm motility and motility character after freezing and thawing. In the present study, we used fertile bulls, but the concentration of intact Lf in the semen was unknown, which might be the reason why the effect of Lf on the fertility rate was not observed to be significant. In future experiments it would be valuable to demonstrate the effect of Lf in subfertile semen.

Comparison between glycerol and ethylene glycol for dog semen cryopreservation

The aim of this study was to compare the effect of ethylene glycol versus glycerol for dog semen freezing, on post-thaw longevity, motility and motility parameters, and on plasma membrane functional integrity. Semen was diluted in two steps with an egg yolk TRIS extender containing a final concentration of either 5% glycerol or 5% ethylene glycol, and frozen in 0.5 ml straws, with 100 × 10 6 spermatozoa/ml, over nitrogen vapours. Semen motility was evaluated both under a light microscope and with a Computer Assisted Motility Analyser System, immediately after thawing and then hourly till 4 h of incubation. Sperm membrane functional integrity was assessed with the hypoosmotic swelling test (60 mOsm fructose solution) applied at thawing and then hourly, for 4 h, on incubated samples. Motility (light microscope) and total and progressive motility (analyser) were significantly higher in ethylene glycol frozen samples at thawing ( P < 0.01); from hour 1 onwards the effect of the cryoprotectant became not significant. Semen frozen with ethylene glycol showed higher path velocity and higher straight line velocity till 3 h after thawing; however, ethylene glycol semen samples also showed higher curvilinear velocity and higher lateral head displacement, which may indicate a capacitation-like condition affecting sperm membranes and possibly reducing post-thaw longevity. Functional integrity of plasma membrane was similar in glycerol and ethylene glycol samples till 3 h after thawing, then ethylene glycol samples showed a higher decline. The strong though short-lived positive effect of ethylene glycol is worth being evaluated further.

Efeito de três diferentes diluidores sobre o sêmen canino submetido a dois protocolos de descongelamento

Com objetivo de avaliar o efeito de dois protocolos de descongelação do sêmen canino congelado em três diluidores foram coletados ejaculados de nove cães. Cada ejaculado foi dividido em 3 amostras, centrifugado e os pellets tratados sob diferentes protocolos: 1º - meio á base de glicina e gema de ovo; 2º - meio á base de TRIS e 3º - meio M9. Foram envasadas 28 palhetas de 0,5ml segundo cada protocolo. Amostras tratadas sob cada protocolo foram avaliadas (M1) quanto a motilidade, vigor e integridade das membranas. As palhetas foram refrigeradas a 5ºC por 60 min. e congeladas em N2. Uma amostra de cada protocolo foi avaliada quanto: motilidade, vigor e integridade das membranas após o equilíbrio (M2). Eram sempre descongeladas três pares de palhetas, sendo que cada par havia sido congelado sob um mesmo protocolo. Uma das palhetas de cada par era descongelada a 37ºC por 30 segundos e outra a 72ºC por 8 segundos. Cada grupo foi avaliado após o descongelamento (M3) quanto a motilidade e vigor espermático, avaliação computadorizada do movimento, integridade das membranas e avaliação acrossomal por meio de Fitc-PNA e PI. As amostras foram mantidas em banho Maria a 37ºC por 1h para o teste de longevidade espermática e reavaliadas (M4) quanto à motilidade e vigor espermáticos, integridade das membranas. Após análise estatística concluímos que as células espermáticas caninas descongeladas a 72ºC por 8 segundos apresentaram um maior somatório de bons resultados quanto aos testes de viabilidade espermática in vitro empregados.

Daya Hidup Spermatozoa Epididimis Domba Garut yang Dikriopreservasi Menggunakan Modifikasi Pengencer Tris

Sperm collected from cauda epididymis is a source of male gametes. The purposes of this study was to evaluate an quality of frozen-thawed sperm of garut ram which was collected from cauda epididymis and cryopreserved with modified Tris extender, i.e: Tris extender (control, KT), Tris extender + 60 mM lactose (LS), and Tris extender + 60 mM lactose + 0.05% glutathione (GL). Quality of collected sperm including concentration, motility, live sperm, abnormality, cytoplasmic droplet, intact acrosomal cap (IAC), and intact plasma membrane (IPM) were evaluated. Results showed that mean of sperm concentration, percentages of motility, live sperm, abnormality, cytoplasmic droplet, IAC, and IPM of fresh epididymal sperm were 13,993.33 million/ml, 70.83, 82.83, 10.83, 8.5, 85.83, and 81.33%, respectively. Sperm quality after equilibration for LS and GL were significantly (P

Ultramicroscopic and biochemical changes in ram spermatozoa cryopreserved with trehalose-based hypertonic extenders

The ability of a range of extenders to cryopreserve ram spermatozoa was tested. The extenders were modified by the inclusion of citrate, Tris buffer, trehalose, and EDTA. Ejaculates from three Pampinta rams were evaluated and pooled at 30 °C. The semen was diluted to contain 1 × 10 9 cells/mL, cooled to 5 °C, loaded into 0.25-mL straws, frozen and stored in liquid nitrogen. Evaluation was based on the hypoosmotic swelling test (HOS test), electron microscopy, and biochemical parameters such as lipid peroxidation and reduced and total glutathione levels, all measured after thawing. The HOS test indicated that the percentage of intact plasma membranes after freezing and thawing was significantly higher for the hypertonic extender containing trehalose (T), compared with an extender containing trehalose + EDTA (TE) or an isotonic Tris extender (B) ( p < 0.05). Membrane evaluation by ultramicroscopy also indicated better sperm cryopreservation in extender T compared with the others, and there was a significant reduction in the number of damaged membranes (27%, p < 0.0002). The level of reduced glutathione was significantly higher after sperm cryopreservation in either hypertonic diluent (T and TE) with respect to the isotonic extender B, immediately after thawing (12%) and after a 3-h post-thawing thermotolerance test at 37 °C (17%, p = 0.007). Total glutathione levels did not show statistical differences among the extenders. After 3 h post-thawing incubation at 37 °C, lipid peroxide levels in spermatozoa were statistically lower for T than TE (35%) or isotonic extender B (44%) ( p = 0.002). Taken together these results indicate a reduction in the oxidative stress provoked by freezing and thawing when semen is cryopreserved in extender T. The antioxidant properties of extender T may be related to its effectiveness in membrane cryopreservation.

Influence of glucose and fructose in the extender during long-term storage of chilled canine semen

The use of chilled, extended semen in dog breeding is becoming increasingly popular as preparation and transportation is less expensive and regulations are often less complicated than for frozen semen. Sugar is one of the main constituents in semen extenders, and glucose and fructose are metabolized in separate pathways by freshly ejaculated dog sperm. In this study, glucose, fructose or an equal mixture of both were used in an egg-yolk–tris (EYT) extender at two different concentrations (10 and 70 mM). EYT extender without sugar supplementation, providing only the glucose (3–4 mM) originating from the egg-yolk, served as a control. The longevity of the chilled semen at 5 °C was 23 days: the quality of physical and functional characteristics decreasing with time. Glucose and fructose had a strong influence on motility and movement patterns of chilled canine semen. The beneficial effect of 70 mM sugar concentrations compared to 10 mM and the control was pronounced, and maintained sperm motility ≥70% for 8 days of storage, compared to for 4 days in the control extender. Fructose maintained higher sperm motility than did glucose and the mixture. VAP values were higher in sugar-supplemented extenders ( P<0.05). Neither type nor concentration of the two sugars influenced sperm plasma membrane, acrosome integrity or the acrosome reaction following ionophore challenge (ARIC). Sugar consumption by dog sperm varied between the different periods of storage and with sugar concentrations provided in the extenders. Glucose consumption by dog sperm was greater than fructose consumption when both sugars were present in equal amounts, indicating that dog sperm used glucose in preference to fructose. In conclusion, the major influence of the two sugars on chilled semen was to support motility. EYT extender supplemented with fructose at a concentration of 70 mM was found to be the best of the tested extenders for long-term preservation of chilled canine semen.

Effect of blood admixture on in vitro survival of chilled and frozen–thawed canine spermatozoa

Hematospermia in the dog usually occurs secondary to benign prostatic hypertrophy or trauma of the penis or prepuce during semen collection. Regarding the difficulty of removing blood cells from a hematospermic sample, the present study was performed to determine whether blood contaminated ejaculates can still be chilled (4 °C) or frozen (−196 °C) without an additional decrease in sperm quality. In the first experiment, blood additions of up to 10% exerted no negative effects on the functional characteristics of canine spermatozoa cooled (4 °C) and stored for 4 days in an egg-yolk–Tris extender. In contrast, in experiment 2, blood admixtures of 4% or more clearly caused negative effects on cryopreserved (−196 °C) spermatozoa, mainly on the motility parameters, on the membrane integrity and on the acrosomal status of the spermatozoa. In experiment 3, we showed that these negative effects of blood admixture on cryopreserved spermatozoa were mainly associated with the red blood cells (RBCs) whereas the addition of plasma, serum or inactivated serum exerted little or no negative effect. Moreover, in experiment 4, we showed that 58.3±11.6% of the RBCs hemolysed after a freeze–thaw process. In experiment 5, a clear and negative effect of hemoglobin on cryopreserved canine spermatozoa was observed. We conclude that the presence of up to 10% blood is not detrimental for the storage of chilled canine spermatozoa and that the detrimental effects of blood on cryopreserved spermatozoa are at least partly attributable to the high amount of hemoglobin originating from the RBC hemolysis observed after freezing and thawing.

Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer

Computerized measuring devices are needed to assess canine semen quality objectively both for research and practical purposes. As internal image settings may influence the results considerably, the effect of different technical settings and semen processing on the parameters assessed by the Hamilton-Thorne Ceros 12.1 semen analyzer (HTR Ceros 12.1) was investigated. The frame rate (15, 30 or 60 frames/s) significantly ( P<0.05) influenced most of the measured motility characteristics in experiment 1 while no differences in the motility parameters were found using a different sampling duration (0.5 or 1 s, i.e. 30 or 60 frames scanned) in experiment 2. In experiment 3, an increase in sperm velocity (VAP, VSL, VCL), in linearity and in the percentage of motile and rapidly moving spermatozoa was observed with increasing sperm concentrations (25×10 6, 50×10 6 or 100×10 6 ml −1). In experiment 4, a clear effect of the diluent used was visible with higher velocity parameters (VAP, VSL, VCL) and higher percentages of motile, progressive and rapid spermatozoa for semen samples diluted in Hepes-TALP or prostatic fluid in comparison with physiological saline or egg-yolk–Tris extender. In experiment 5, significant ( P<0.01) and high correlations were found between the conventional dog semen analysis methods and HTR Ceros 12.1 measurements ( n=97 semen samples) for the sperm concentration ( r=0.91), the motility ( r=0.74) and the progressive motility ( r=0.84). In experiment 6, the ejaculates from 21 proven, fertile dogs were compared with the ejaculates of a population ( n: 11) of young beagles (1.5 years) but no significant differences in HTR Ceros 12.1 measurements were found between the two groups. Based on our results, diluting dog semen samples to 50×10 6 ml −1 with physiological saline solution and scanning 30 frames at a frame rate of 60 frames/s (i.e. a scanning time of 0.5 s), are the set-up parameters proposed to obtain objective and standardized canine semen motility results using the HTR Ceros 12.1.

Effectivity of various concentration of lactose in Tris extender on liquid semen viability of Saanen bucks

The objective of this study is to determine the best concentration of lactose in Tris extender in maintaining liquid semen viability of Saanen bucks stored at 5°C. Four heads of Saanen bucks of 2-4 years old were used as semen source. Semen was collected once a week using an artificial vagina. Variance analysis was conducted based on completely randomized design with three treatments, i.e 30, 60, and 90 mM lactose (L30, L68 and L90). Duncan test was used to observe the different between treatment. Results indicated that the mean percentage of motility, live sperm, intact plasma membrane, and intact acrosomal cap of liquid semen in treatment L30 (70.83; 78.20; 79.56 and 81.20% respectively) were significantly higher (P<0.05) than treatment L60 (67.08; 73.73; 75.01 and 76.87% respectively) and treatment L90 (61.50; 71.88; 69.08 and 71.33% respectively). After storage for 48 hours at 5°C apparently the mean percentage of motility, live sperm, intact plasma membrane and intact acrosomal cap in treatment L30 were significantly higher (P<0.05) than those of in treatment L60 and L90. Decreasing percentage of motility, live sperm, intact plasma membrane, and intact acrosomal cap during storage at 50C from 0 until 48 hour in treatment L30 (42.83; 19.75; 40.84 and 39.00% respectively) were significantly lower (P<0.05) than those of in treatment L60 (55.41; 23.06; 50.14; and 50.90% respectively) and treatment L90 (54.83; 29.93; 50.14 and 50.97% respectively). It was concluded that supplementation 30 mM lactose in Tris extender could maintain liquid semen viability in Saanen bucks. Key words: Saanen bucks, lactose, liquid semen