Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post-thaw longevity and in vitro fertility.

BACKGROUND: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. OBJECTIVES: To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. MATERIALS AND METHODS: At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4 h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t0 ), and after incubation (t4 ). The in vitro fertility of the spermatozoa was assessed at t0 and t4 . RESULTS: For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3 cm above LN2 and thawed at 50 and 65°C (P < 0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t0 , as well as higher FMI, total and progressive motility, and linearity at t4 (P < 0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P < 0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. CONCLUSION: Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

Effect of cholesterol supplementation on cryosurvival of goat spermatozoa.

AIM: Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen. MATERIALS AND METHODS: A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 10(6) spermatozoa, respectively, and Group IV, treated with 1 mg methyl-ß-cyclodextrin (MßCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa. RESULTS: Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 10(6) spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 10(6) spermatozoa. In general, MßCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h. CONCLUSION: Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.

Cysteine addition on short-term cooled boar semen preservation and its relationship with swine field fertility / Adição de cisteína na preservação de sêmen suíno refrigerado e sua relação com a fertilidade suína

Artificial insemination is routinely used in the swine industry to reduce the costs of production through to increase the efficiency of the refrigerated boar semen process. The objective of this study was to evaluate the effect of different levels of cysteine (CYS) added to the Beltsville Thawing Solution (BTS) extender semen during cooling for up to 72 hours. Ejaculated from three boars were collected with the gloved-hand technique and semen aliquots were diluted in BTS as follow: BTS only (BTS), BTS + 0.1mM cysteine (CYS0.1), BTS + 0.5mM cysteine (CYS0.5), BTS + 1.0mM cysteine (CYS1.0), BTS + 2.5mM cysteine (CYS2.5), BTS + 5.0mM cysteine (CYS5.0), BTS + 10.0mM cysteine (CYS10.0), and BTS + 20.0mM cysteine (CYS20.0). Evaluation of sperm integrity were analyzed using 0.5mg/ml propidium iodide (plasma membrane), 100µg/ml isothiocynate-conjugated Pisum sativun agglutinin (acrosomal membrane) and 153µM 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (mitochondria potential) after semen dilution at specific times (0, 24, 48 and 72 hours). Additionally, we also evaluated the effects of 5.0 mM CYS addition in the BTS extender on the maintenance of sperm quality and their influence on fertility in the swine production. After artificial insemination, animals were evaluated based on the estrous return and the number of piglet's born. Cysteine at concentrations of 10.0 and 20.0mM resulted in more pronounced reductions even at the time zero. Semen viability decreased to levels below 10 percent at these high levels of CYS in the first 24 hour of storage at 17ºC. At the end of the storage time, less than 65 percent of sperm cells had intact plasma membrane in all groups. The sperm viability decreased significantly when the semen was added at high concentrations of CYS (time "0"; CYS10.0 and CYS20.0; p<0.05), when compared to the other CYS concentrations. The BTS (10.20±0.39) treated group showed a lower rate of estrus return when compared to other (BTSCYS; 86.05±039), and it showed also the highest total number of piglets borne per treatment (12.71±3.38 vs. 9.00±3.38, respectively). In conclusion, the addition of CYS in the BTS semen extender did not maintain spermatic viability of boar cooled spermatozoa and it results in a higher percentage of return to estrus and lower number of piglets borne.

A inseminação artificial é usada rotineiramente na indústria suinícula para reduzir os custos de produção além de obter maior eficiência reprodutiva durante o processo de resfriamento do sêmen. O objetivo deste trabalho foi avaliar o efeito da adição de diferentes concentrações de cisteína (CIS) ao diluidor de sêmen Beltsville Thawing Solution (BTS) resfriado sobre a qualidade espermática por até 72 horas. Foram coletados ejaculados de três cachaços e as amostras de sêmen foram diluídas em BTS, conforme os seguintes tratamentos: BTS (grupo controle); CIS0,1 (BTS + 0,1mM de cisteína); CIS0,5 (BTS + 0,5mM de cisteína); CIS1,0 (BTS + 1,0mM de cisteína); CIS2,5 (BTS + 2,5mM de cisteína); CIS5,0 (BTS + 5,0mM de cisteína); CIS10,0 (BTS + 10,0mM de cisteína) e CIS20,0 (BTS + 20,0mM de cisteína). A avaliação da integridade espermática foi determinada através de sondas fluorescentes em uma combinação de 100µg/mL FICT-PSA (isotiocinato de lecitina), 0.5mg/ml PI (iodeto de propidio), e 153µM JC-1 (5,5',6,6'-tetracloro-1,1',3,3'-tetraetillbenzimidazolil iodeto de carbocianina). As avaliações dos tratamentos foram realizadas 0, 24, 48 e 72 horas após a diluição do sêmen. Adicionalmente, foi avaliado o efeito da adição de 5,0 mM de cisteína ao diluidor BTS na manutenção da qualidade espermática e no efeito na fertilidade em suínos. Após a inseminação artificial, as fêmeas foram avaliadas quanto a taxa de retorno e o tamanho da leitegada. Durante todos os períodos analizados, os grupos CIS10,0 e CIS20,0 apresentaram menor número de espermatozóides viáveis em relação aos demais grupos. A viabilidade espermática diminuiu a níveis abaixo de 10 por cento nos tratamentos CIS10,0 e CIS20,0 nas primeiras 24 horas de armazenamento a 17ºC. Ao final do período de armazenamento todos os grupos apresentavam média inferior a 65 por cento de espermatozóides com a membrana plasmática intacta. A viabilidade espermática diminuiu significativamente quando altas concentrações de CIS (hora "0"; CIS10,0 e CIS20,0; p<0.05) foram adicionadas ao sêmen comparadas com as demais concentrações. O grupo BTS (10,20±0,39) apresentou menor taxa de retorno ao estro comparado com BTSCIS (86,05±0,39), além de apresentar maior número de leitões nascidos (12,71±3,38 vs . 9,00±3,38, respectivamente). Portanto, podemos concluir que a adição de CIS ao diluidor BTS não mantém a qualidade espermática e resulta em maior taxa de retorno ao estro e menor número de leitões nascidos.