Efeito de vitaminas adicionadas ao diluente ACP-104 sobre a qualidade do sêmen criopreservado de carpa comum (Cyprinus carpio) / Effect of vitamins added to the ACP-104 extender on the quality of cryopreserved semen of common carp (Cyprinus carpio)

O objetivo da presente pesquisa foi alcançado com a divisão da pesquisa em dois experimentos: (1) aperfeiçoar o protocolo de congelação utilizando água de coco em pó (ACP-104) como diluente para a criopreservação seminal de carpa comum; (2) avaliar o efeito da suplementação das vitaminas C (ácido ascórbico) ou E (α-tocoferol) sobre os melhores diluidores testados no experimento 1 na qualidade do sêmen pós-descongelado da espécie. Para o experimento 1, foram formados oito pools de sêmen, provenientes de 14 machos selecionados. As amostras seminais coletadas foram avaliadas quanto à motilidade total, à velocidade, ao percentual de espermatozoides normais e à vitalidade espermática antes e depois da criopreservação seminal. Esta foi realizada em meio ACP-104 acrescido de dimetilsulfóxido (DMSO), ou etilenoglicol (EG), ou glicerol, ou metanol, todos à concentração de 10%, diluídos em 1:3 (sêmen:diluidor). As amostras foram, então, congeladas em vapor de nitrogênio líquido em dry shipper e estocadas em nitrogênio líquido (-196°C). Para o experimento 2, foram formados oito pools provenientes da coleta de sêmen de 15 machos. As amostras seminais foram avaliadas seguindo as mesmas análises do experimento 1, acrescentando-se a duração da motilidade total. A criopreservação seminal utilizou-se do meio ACP-104 acrescido de DMSO ou EG, suplementado ou não com vitamina C ou E. Os melhores resultados encontrados no experimento 1 foram obtidos com o DMSO e o EG. Estes não diferiram significativamente entre si para a motilidade total (24% e 28%; P>0,05) e a normalidade espermática (32% e 26%; P>0,05), respectivamente. Para o experimento 2, o EG suplementado com vitamina E produziu significativamente resultados superiores de motilidade total, normalidade espermática e duração da motilidade em relação ao DMSO, concluindo-se que o EG deve ser, portanto, o crioprotetor de escolha a ser utilizado com o ACP-104 suplementado ou não com vitamina E.(AU)

The objective was achieved by dividing the research into two experiments: (1) improving the freezing protocol using powdered coconut water (ACP-104) as a diluent for the cryopreservation seminal of common carp; (2) evaluating the effect of supplementation of vitamins C (ascorbic acid) or vitamin E (α-tocoferol) with the best extenders tested in experiment 1 on the quality of post-thawed. For experiment 1, semen pools from 14 selected males were formed. Seminal samples were evaluated for total motility, velocity, percentage of normal sperm and sperm vitality before and after the seminal cryopreservation. This was done in ACP-104 extender plus dimethyl sulfoxide (DMSO), or ethylene glycol (EG), or glycerol or methanol all at concentration 10% diluted in 1:3 (semen:extender). The samples were frozen in vapors of nitrogen into dry shippers and stored in liquid nitrogen (-196 °C). For experiment 2, eight pools were formed from the 15 males. The semen samples were evaluated following the same analysis of experiment 1 adding duration of total motility. The sperm cryopreservation was performed in extenders ACP-104 plus DMSO or EG supplemented or not with vitamin C or E. The best results found in Experiment 1 were obtained with DMSO and EG. They do not differ significantly for total motility (24% and 28%; P>0.05) and normal sperm (32% and 26%; P>0.05) respectively. For experiment 2, EG supplemented with vitamin E, produced significantly better results overall motility, sperm normality and duration of motility relative to DMSO. In conclusion, EG should be the cryoprotectant of choice for use with the ACP-104 supplemented or not with vitamin E.(AU)

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen.

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.

Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen.

The objective was to evaluate the effects of various antioxidants and duration of pre-freezing equilibration on cryopreservation of ram semen. Semen samples from four rams were pooled, diluted with Tris-egg yolk extender without antioxidants (control), or supplemented with reduced glutathione (GSH: 0.5, 1.0, and 2.0 mM), superoxide dismutase (SOD: 5, 10, and 20 U/mL), or catalase (CAT: 5, 10, and 20 U/mL), and cryopreserved, immediately after thermal equilibrium was reached at 5 °C (0 h), or 12 or 24 h after equilibration. Total antioxidant capacity was determined in the in natura extenders and after addition of semen samples for various durations of processing (fresh/dilute, throughout refrigeration, and post-thaw). Plasma membrane (PI-CFDA), acrosome integrity (FITC-PNA), and mitochondrial membrane potential (JC-1) were determined in fresh/diluted, refrigerated, and post-thaw samples. Post-thaw sperm motility was assessed with a computerized analysis system (CASA). There were no significant differences in acrosome damage or mitochondrial membrane potential after refrigeration and freeze-thaw, regardless of antioxidant addition. Sperm plasma membrane integrity was worse (P < 0.05) with cryopreservation immediately after equilibration (average 20.1 ± 8.3; mean ± SD) than after 12 h of equilibration (average 42.5 ± 10.9); however, the addition of SOD and CAT (10 and 20 U/mL) resulted in no significant difference between post-equilibration intervals of 0 and 12 h. Total antioxidant activity was not different (P > 0.05) among treatments after sperm addition or throughout the refrigeration and post-thaw. In conclusion, adding GSH, SOD or CAT did not increase the total antioxidant capacity of semen, nor did it enhance the quality of the post-thaw sperm. However, maintenance of ram semen at 5 °C for 12 h prior to cryopreservation reduced membrane damage of frozen-thawed sperm.

In vitro and in vivo evaluation of ram sperm frozen in tris egg-yolk and supplemented with superoxide dismutase and reduced glutathione.

The aim of the present study was to evaluate the in vitro and in vivo effect of the addition of superoxide dismutase (SOD) and reduced glutathione (GSH) to ram semen freezing extender. Significant differences (p < 0.05) were detected between groups regarding total motility (TM), straightness (STR) and wobble (WOB), for which the GSH 7 mM group had lesser TM and better STR than the other groups and the GSH 5 and 7 mM groups had higher wobble values than the control, SOD 25 and 100 U/ml groups. The ultrastructural analysis revealed that the acrosome was better preserved after freezing in the SOD 100 U/ml and GSH 2 and 5 mM (p < 0.05) groups than the other groups, whereas mitochondria in both the control group and the 7 mM GSH group suffered the greatest damage. The plasma membrane remained preserved after freezing, regardless of the group. For in vivo fertilization, the SOD group achieved better results than the GSH group (p > 0.05). It can therefore be concluded that the addition of SOD 100 U/ml and GSH 2 and 5 mM preserves the acrosome integrity of frozen ram spermatozoa, while the addition of SOD 100 U/ml to Tris egg-yolk extender offers protection to the membranes of sperm cells after thawing.

The effects of chronic cannabis treatment upon brain 5-hydroxytryptamine, plasma corticosterone and aggressive behavior in female rats with different hormonal status.

Ovariectomized rats, chronically treated with cannabis extract or control solution, were given different hormonal treatments. Results indicated that both cannabis-treated and estrogen-treated animals were more aggressive than controls. Furthermore, aggressiveness was virtually abolished when cannabis-treated females were made sexually receptive by estrogen and progesterone treatments. After 25 days of cannabis or control solution treatment, all subjects were sacrificed. The levels and turnover rate of brain 5-HT and peripheral plasma corticosterone were then assayed. Data indicated both a significant inverse relationship between plasma corticosterone and whole brain levels of 5-HT(r = -0.742 to -0.985) for all groups and a significant positive relationship between aggressive behavior and plasma corticosterone (r = +0.675 to +0.946) in all groups that were fighting prior to decapitation. Results are tentatively explained, suggesting that the variability of the female response to stress during the different phases of the estrus cycle, permitted them to perform differently after chronic cannabis treatment. 5-HT is apparently involved, either directly in its effects on aggressive behavior or indirectly through the pituitary-adrenocortical axis activation.