Effect of Amide on Semen Cryopreservation of Curimba (Prochilodus lineatus).

BACKGROUND: The low molecular weight and high cellular permeability of amides make them suitable for use as penetrative cryoprotectants for sperm cells. OBJECTIVE: This study aims to evaluate the effect of dimethylformamide (DMF) and dimethylacetamide (DMA) on sperm cryopreservation of Curimba (Prochilodus lineatus). MATERIALS AND METHODS: Semen samples were diluted in media containing cryoprotectants [DMF, DMA and dimethyl sulfoxide (DMSO)]. Parameters of motility, membrane integrity, DNA integrity, mitochondrial functionality, viability and fertility were assessed upon thawing. RESULTS: As compared to the 10% DMSO, DMA at 5% and DMF at 2% obtained the best results for the integrity of membrane, DNA and mitochondria; the motility parameters were best in the 2% and 5% DMF treatments. The best fertilization rates were demonstrated in 2%, 5%, and 8% DMF treatment groups. CONCLUSION: DMF at 2%, 5%, and 8% provided the best results for both in vitro and in vivo assessments, and can efficiently cryopreserve semen of Prochilodus lineatus.

Exogenous ATP in the Cryopreservation of Brycon orbignyanus Spermatozoa.

BACKGROUND: ATP exogenous (ATPe) has been used successfully in improving motility and fertility for many animal species. However this has not yet been tested on Brycon orbignyamus. OBJECTIVE: The objective of this study was to evaluate the use of ATPe for the cryopreservation of sperm from B. orbignyamus. MATERIALS AND METHODS: The ATPe concentrations tested were 1.0 µM, 5.0 µM and 10 µM combined with Beltsville Thawing SolutionTM extender and dimethylformamide at 7.5%. The sperm were frozen in a nitrogen vapour vessel and stored in liquid nitrogen at -196 ºC. The parameters of viability post-thawing were evaluated using CASA, and flow cytometer. RESULTS: The ATPe did not promote improvements in spermatic kinetics, and in the higher concentrations caused a worsening in these parameters. Also there was loss of mitochondrial functionality and greater cellular disruption with the concentration of 10 µM. CONCLUSION: We do not recommend the addition of ATP for cryopreserving B. orbignyamus.

Impact of Distinct Freezing Curves on the Quality of Ram Sperm after Thawing.

BACKGROUND: Automated equipment with customized freezing curves can be used to cryopreserve ram sperm, but none is considered a standard. OBJECTIVE: This study compared the post-thawing quality of ram sperm frozen using a conventional freezing curve and two controlled-rate curves. MATERIALS AND METHODS: Six ejaculates were collected from four rams (n = 24). In the conventional curve (110 min), sperm was cooled at -0.3 to -0.5°C min-1 until 5°C, stabilized for 60 min and exposed to liquid nitrogen (LN2) vapor for 10 min (-80°C) before submersion. The slow-customized (SC) curve (126.2 min) used a rate of -0.25°C min-1 until 5°C, stabilization for 60 min and a rate of -20°C min-1 until -120°C before immersion in LN2. Rates for the fast-customized (FC) curve (75 min) were: -0.3°C min-1 until 5°C; -3°C min-1 until -10°C; -5°C min-1 until -35°C; and -4°C min-1 until immersion in LN2 (-43°C). RESULTS: Velocity in a straight line and beat-cross frequency were lower for spermatozoa frozen with the FC than with the conventional curve (P < 0.05). The FC curve resulted in more membrane and acrosome damages than the other curves (P < 0.05). Mitochondrial membrane potential was lower with the SC than with the other curves (P < 0.05). CONCLUSION: The conventional curve was more efficient than both tested automated freezing curves. The FC curve may be an alternative to the SC curve due to the shorter processing time.

The effects of xanthan gum on equine sperm quality during cooling storage / Efeitos de xantam gum em qualidade de esperma equino durante armazenamento resfriado

This study was designed to evaluate the possible benefits of adding xanthan gum to a standard extender for equine through in vitro analyzes of sperm quality. Semen was collected four times from five different stallions (n= 20 samples) and subjected to cooled storage under different conditions: control (only standard extender) and three different concentrations of xanthan gum (0.01%, 0.12%, and 0.25%) supplemented to the extenders. Sperm parameters, such as motility, mitochondrial functionality, and membrane, acrosome, and DNA integrity were measured after 0h, 24h, 48h, and 72h of sperm storage at 5ºC. Our observations indicated that sperm motility declined with longer cooling period with the 0.25% xanthan gum supplementation group compared with the control group. Other parameters, such as mitochondrial functionality and membrane and acrosome integrity also declined for all treatments during storage; however, no differences were observed between xanthan gum and control groups. DNA integrity did not significantly change during the storage. In conclusion, the addition of xanthan gum to equine semen extender is not harmful to the sperm structure, despite reducing the sperm motility.(AU)

Esse estudo foi desenvolvido para avaliar os possíveis benefícios de acrescentar xanthan gum a um extensor padrão através de analises in vitro de qualidade de esperma. Semen foi coletado quatro vezes de cinco garanhões diferentes (n = 20 amostras) e submetido a armazenamen to resfriado em diferentes condições: controle (apenas extensor padrão) e três diferentes concentrações de xanthan gum (0,01%, 0,12% e 0,25%) suplementado aos extensores. Parâmetros dos espermatozoides, como mobilidade, funcionamento mitocondrial e integridade de membranas, acrossomos e DNA forma medidos após 0h, 24h, 48h e 72h de armazenamento a 5oC. Nossas observações indicaram que motilidade reduziu com armazenamento resfriado prolongado no grupo de 0,25% de suplementação de xanthan gum comparado ao grupo controle. Outros parâmetros, como funcionalidade mitocondrial e integridade de membrana e acrossomos também reduziu em todos os tratamentos durante o armazenamento, no entanto não foram detectadas diferenças significativas entre grupos tratados e grupo controle. Integridade de DNA não mudou significativamente durante armazenamento. Em conclusão, a adição de xanthan gum a extensor de sêmen equino não é danosa à estrutura do espermatozoide apesar de reduzir motilidade.(AU)

Boar sperm quality after supplementation of diets with omega-3 polyunsaturated fatty acids extracted from microalgae.

This study evaluated effects of diet supplementation with omega-3 polyunsaturated fatty acids (PUFA) from microalgae on boar sperm quality. Two groups of boars (n = 3 each) were fed during 75 days either a commercial diet (control), or the same diet supplemented with omega-3 PUFA from the heterotrophic microalgae Schizochytrium sp. (120 g/kg). Sixteen ejaculates were collected per boar. Some sperm kinetics parameters were inferior for supplemented than for control boars (p < .05): distance average path; distance in both curved and straight line; velocity average path, velocity in both curved and straight line; and amplitude of lateral head displacement. Spermatozoa from supplemented boars presented lower mitochondrial functionality, but greater membrane fluidity compared to the control group (p < .01). Membrane and acrosome integrity, production of reactive oxygen species and lipid peroxidation did not differ (p > .05). Serum cholesterol levels were greater (p < .05) for supplemented than for control boars at the 30th and 60th d of supplementation, but levels of triglycerides and IGF-1 did not differ (p > .05). Compared to the control, spermatozoa of supplemented boars were slower, travelled shorter distances and presented impaired energy metabolism, but their greater membrane fluidity may potentially favour their cryopreservation.

Presence of leptin and its receptor in the hypothalamus, uterus and ovaries of Swine females culled with distinct ovarian statuses and parities.

Leptin acts on energy metabolism, affecting reproductive functions through activation of its receptors in the brain and in reproductive organs. This study compared the presence of leptin and its receptor (ObR-b) in hypothalamus neurons, endometrial glands and oocytes of culled swine females across ovarian statuses and parities. Immunohistochemistry was done in samples of uterus, ovaries and hypothalamus from 28 culled females, using polyclonal antibodies antileptin and ObR-b. Immunolabelling was compared for sows categorized by parity at culling (0, 1, 2-4 and <4) and ovarian status (luteal and follicular phases of the oestrous cycle and with cysts). Immunolabelling for leptin and ObR-b in neurons and oocytes was weaker in females with cysts (p < 0.05) than in those at the follicular phase. In endometrial glands, leptin immunolabelling was less intense in females with cysts (p < 0.05), but immunolabelling for ObR-b was similar across ovarian statuses (p > 0.05). In sows culled with 2-4 parities, leptin immunolabelling in neurons and endometrial glands was more intense than in nulliparous females (p < 0.05). In comparison with sows culled at greater parities, ObR-b immunolabelling for nulliparous females was less intense in endometrial glands and in oocytes (p < 0.05), but more intense in neurons (p < 0.05). Thus, in swine, the presence of leptin and ObR-b varies across parities and is more intense in the uterus, ovaries and hypothalamus of females that were cycling before culling than in those having cystic ovaries.

Use of amides as cryoprotectants in extenders for frozen sperm of tambaqui, Colossoma macropomum.

Amides were tested as cryoprotectants in comparison with glycerol and DMSO (more traditional cryoprotectants) for recovery of Colossoma macropomum (tambaqui fish) sperm. Milt was extended in Beltsville Thawing Solution, then frozen with the addition of 2%, 5%, 8%, or 11% of: (1) dimethylacetamide (DMA), (2) dimethylformamide (DMF), (3) methylformamide (MF), or with 5% glycerol or 10% dimethylsulfoxide. Fertilization rates were greatest (P<0.001) with amides; 8% DMF (91.6±1.3%), 5% DMF (88.9±1.6%), and 8% MF (83.0±1.6%), which did not significantly differ among themselves, when compared with glycerol (51.6±2.4%) and DMSO (61.9±3.1%). The best hatching rates (P<0.001) also occurred for 5% or 8% DMF and 8% MF (79.1±3.1, 87.6±1.5, and 74.8±3.0, respectively) and were also similar (P>0.05). For such treatments, both fertilization and hatching rates were similar (P>0.05) to those with fresh sperm (91.7±1.4 and 87.4±1.4, respectively). The best sperm motility across extenders (at least 55.7%) was with 5%, 8%, and 11% DMF (P<0.001). Those same treatments, along with 11% MF, provided the longest (P<0.001) period of motility (at least 1 min). The greatest sperm integrity (more than 54%) was with 5% and 11% MF and with DMA and DMF at all tested concentrations (P<0.001). The greatest (P<0.001) sperm viability (at least 31%) was for 5%, 8%, and 11% DMA, and with 8% and 11% MF, and also for DMF at all tested concentrations. Sperm DNA integrity was best (more than 50%) for 2%, 5%, and 8% MF and for DMA and DMF at all concentrations (P<0.001), whereas 2% DMA, 11% MF, 11% DMF, and the three amides at both 5% and 8% yielded the highest mitochondrial functionality (at least 44%; P<0.001); thus, 8% MF and both 5% and 8% DMF were the cryoprotectants with the best postthaw quality for C. macropomum sperm.