Optimizing semen cryopreservation in Calomys laucha: a step forward in rodent reproductive research.

BACKGROUND: Examining semen cryopreservation in Calomys laucha offers valuable insights for reproductive research and species conservation. OBJECTIVE: To determine the most effective sugar for the cryopreservation of C. laucha semen. MATERIALS AND METHODS: Using 36 epididymides from C. laucha, semen samples were diluted in a 3% skimmed milk medium supplemented with one of four sugars (glucose, fructose, lactose, or sucrose) at a concentration of 0.3 M. These mixtures underwent a conditioning phase at 37 degree C for 10 min, cooled to -80 degree C for another 10 min, and were subsequently stored in liquid nitrogen. RESULTS: Upon thawing, samples treated with lactose and glucose solutions show superior sperm motility, achieving 8.2% and 10.0% respectively, in contrast to the fructose (2.0%) and sucrose (4.1%) mixtures. Furthermore, samples preserved in glucose registered the highest sperm penetration rates, reaching 44.9%. CONCLUSION: Our findings suggest that a cryopreservation medium containing 0.3 M glucose can contribute to the safeguarding C. laucha rodent semen. https://doi.org/10.54680/fr24210110612.

Freezing of equine semen is influenced by exposure time and concentration of the cryoprotectant glycerol.

BACKGROUND: Glycerol is a cryoprotectant widely used in the freezing of mammalian semen, but no study has demonstrated its optimum concentration and the appropriate exposure time for equine species. OBJECTIVE: To demonstrate that the exposure time (15, 30, 45, 60, 75 and 90 min) versus concentration (2, 3, 4 and 5%) of the cryoprotectant glycerol influences the freezing success of equine semen. MATERIALS AND METHODS: The ejaculate of 12 stallions were frozen in different glycerol concentrations following different exposure times. The thawed sperm was evaluated for kinetic parameters using a Computer Assisted Semen Analysis (CASA) system and cell feature parameters were assessed by flow cytometry. RESULTS: Considering the total and progressive motility of the spermatozoa, we concluded that protocols using 5% glycerol for 15 and 30 min exposure, 4% glycerol for 45 min exposure and 3% glycerol for 90 min exposure generated the best results. CONCLUSION: We suggest the use of any of these protocols for a better cryopreservation of equine semen. Doi: 10.54680/fr23410110412.

Trehalose in extenders for cryopreservation of Tambaqui (Colossoma macropomum) sperm.

BACKGROUND: Sugars may act as either energy substrates or non-penetrating cryoprotectants. OBJECTIVE: Inclusion of non-penetrating trehalose was tested in extenders for the cryopreservation of Tambaqui (Colossoma macropomum) sperm. MATERIALS AND METHODS: Sperm was extended 1/9 (v/v) in Beltsville Thawing Solution (BTS) with 10% DMSO (control) or 50, 100, 150 and 200 mM trehalose without 10% DMSO. Post-thawed sperm quality was evaluated, including fertilization and hatching rates, sperm motility, motility period and viability, integrity of sperm membrane and DNA, and mitochondrial functionality. RESULTS: Extenders with 100 - 150 mM trehalose achieved fertilization and hatching rates similar to those of the 10% DMSO-treated sperm samples. Trehalose at 100 and 150 mM provides better protection than 10% DMSO treatment for sperm motility, viability, DNA integrity and mitochondrial functionality. Fertilization and hatching rates were highly correlated (r = 0.95, P < 0.001). CONCLUSION: The addition of 100 - 150 mM trehalose in extender can replace 10% DMSO for the cryopreservation of C. macropomum sperm. doi.org/10.54680/fr22510110312.

Dimethylacetamide in Rooster Semen Cryopreservation.

BACKGROUND: Sperm cryopreservation of cockerels is a major challenge, and so far there is no adequate information to enable commercial use of frozen semen. OBJECTIVE: To test the toxicity of dimethylacetamide (DMA). MATERIALS AND METHODS: DMA was added at 3%, 6%, 9% and 12% to the freezing diluent, and maintained for equilibration with the semen sample for 1 min, 3 min, 5 min, 7 min and 9 min prior to freezing. Thawed semen was evaluated for kinetic characteristics by computer-assisted semen analysis (CASA) and for structural and functional properties by flow cytometry (plasma membrane rupture, mitochondrial functionality and plasma membrane functionality). RESULTS AND CONCLUSION: The addition of 6% DMA for 3-min equilibration resulted in the highest total and progressive motility, 42.0% and 36.9%, respectively. The point of intersection between a good protection and low plasma membrane rupture was obtained with the addition of 6% of DMA for 3-min equilibration with the rooster semen.

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.

Liquid Storage of Geophagus brasiliensis semen in the Presence of Different Extenders.

BACKGROUND: In order to preserve the genetic diversity of cichlid fish in gene banks, it is necessary to use certain extenders to maintain the integrity of spermatozoa cells during cooling. OBJECTIVE: To evaluate the effects of different extenders on the quality parameters of cooled semen of Geophagus brasiliensis. MATERIALS AND METHODS: Semen samples were collected from seven adult fish and diluted with five extenders: Beltsville Thawing Solution (BTS™), Hanks' Balanced Salt Solution (HBSS), Tris-glucose, Ginsburg's Fish Ringers, and Phosphate buffered Saline. All parameters were evaluated in fresh semen samples and after cooling at 4°C at 0, 24, 48, and 96 h to evaluate cell viability (membrane integrity, DNA, and mitochondrial functionality) and motility rate and weather motility. RESULTS: The BTS and Tris-glucose resulted in the best outcomes (P<0.05) in terms of membrane integrity assessments (35.1% and 30.9%, respectively), DNA integrity (71.6%; 75.7%), mitochondrial function (26.9%; 28.0%) and motility rate (8.6%; 8.6%), respectively, for semen cooled to 4°C for 96 h. However, the 48-h period motility after cooling in BTS was superior to all other treatments. CONCLUSION: BTS and Tris-glucose can be considered as the best extenders for the cold storage of Geophagus brasiliensis spermatozoa.

Effects of Bisphenol A on redox balance in red blood and sperm cells and spermatic quality in zebrafish Danio rerio.

Bisphenol-A (BPA) is a potential endocrine disruptor besides being associated with oxidative damage in several vertebrate classes. In the present study we investigated oxidative effects in erythrocytes and sperm cells as well as spermatic quality in Danio rerio exposed to 14 days at BPA concentrations of 2, 10 and 100 µg/L. Organelles structure, reactive species of oxygen (ROS) and lipoperoxidation (LPO) on erythrocytes and sperm cells were measured by flow cytometry and spermatic parameters were analyzed by the computer-assisted sperm analysis (CASA) system. For both cell types, when compared with control BPA treatment induced a significant increase in ROS and LPO production causing the membrane fluidity disorder, loss of membrane integrity and mitochondrial functionality. Furthermore, it was found a significant increase in DNA fragmentation in erythrocytes of zebrafish BPA exposed. Regarding the spermatic quality, results showed lower sperm motility in animals exposed to BPA, and alterations on velocity parameters of spermatozoa. Thus, the present study concludes that BPA affects the oxidative balance of both cell types, and that can directly affects the reproductive success of the adult Danio rerio. The sensitivity of erythrocytes to oxidative damage induced by BPA was similar to sperm cells, indicating a potential use of blood cells as indicators of oxidative damage present in fish sperm.

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.

Association Between DMSO and Sugars in the Sperm Cryopreservation of Pacu.

　　BACKGROUND: The cryopreservation protocol that has been developed exclusively for the preservation of the sperm of the species different. OBJECTIVE: this study was to evaluate the effect of the association of 10% DMSO with trehalose, raffinose, sucrose and lactose concentrations on the sperm cells of Piaractus mesopotamicus. MATERIALS AND METHODS: Sperms were collected from the animals through abdominal massage. The samples were diluted in the Beltsville Thawing Solution without different concentrations of other sugars (test conditions). Sixty days after the cryopreservation, cell movement analysis was performed using CASA. RESULTS: The results revealed that the parameters for total motility and motility period were superior when 100mM raffinose (P <0.05). The lateral displacement of the head was observed to be improved was 100mM lactose, 150mM sucrose and 150mM raffinose (P <0.05) as compared to treatment wherein lactose (0mM) was omitted. CONCLUSION: the results of our study indicated that the ideal parameters for cryopreservation, were obtained when the cryopreservation fluid contained 100mM raffinose in association with DMSO.

Cooling strategies for brazilian flounder Paralichthys orbignyanus embryos.

BACKGROUND: Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. OBJECTIVE: To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). MATERIALS AND METHODS: Control embryos were maintained at 23 degree C and treated embryos were cooled to 15 degree C, 10 degree C and 5 degree C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). RESULTS: The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. CONCLUSION: As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5 degree C for 30 min are recommended.

Antioxidant Effect of Xanthan Gum on Ram Sperm after Freezing and Thawing.

BACKGROUND: Xanthan gum is used as thickener in media to preserve food products, having cryoprotectant and antioxidant properties that may be relevant for sperm cryopreservation. OBJECTIVE: To evaluate the effects of adding xanthan gum to freezing extenders on post-thawing quality and oxidant activity of ram sperm. METHODS: Ejaculates from seven rams extended TRIS-egg yolk-glycerol were split in three treatments including xanthan gum (0.15%; 0.20%; and 0.25%) and a control with no xanthan gum. RESULTS: After thawing, motility and production of reactive oxygen species (ROS) with 0.20% and 0.25% xanthan gum were lower than for the control (P < 0.05), but mitochondrial functionality and integrity of membrane, acrosome and DNA did not differ (P > 0.05). Xanthan gum at 0.20% and 0.25% may be an efficient antioxidant for frozen-thawed ram sperm, due to the reduction in ROS production.

Quercetin in equine frozen semen.

　　BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. OBJECTIVE: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. MATERIALS AND METHODS: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. RESULTS: The statistical analysis did not reveal any significant modification of seminal parameters. CONCLUSION: Within the concentrations tested, quercetin supplementation in equine freezing extender did not affect progressive motility, mitochondrial functionality, acrosome reaction, membrane integrity or DNA fragmentation index in post-thaw equine semen.

Effect of egg yolk plasma on dog sperm cryopreservation.

This study evaluated the quality of frozen-thawed dog spermatozoon after the inclusion of egg yolk plasma (EYP) instead of whole egg yolk (EY) in the cryopreservation extender and after distinct periods of exposure to EYP. Seven mongrel dogs were used as sperm donors, and EYP was obtained by centrifugation. In Experiment 1, post-thawing sperm motility (MOT) and integrity of membrane (INT) and acrosome (ACR) were superior for spermatozoon extended with 20% EYP T2 than with 20% EY (P < 0.05), although normal sperm morphology (MOR) did not differ (P > 0.05). In Experiment 2, after ejaculates extended with 20% EYP were cooled at 5°C for 2, 6 and 10 h before freezing, MOT, INT and ACR were similar among periods (P > 0.05). Thus, dog spermatozoon extended with 20% EYP can be kept cooled for up to 10 h prior to freezing, achieving post-thawing quality greater than that obtained with the inclusion of EY in freezing extenders.

Contribution of boars to reproductive performance and paternity after homospermic and heterospermic artificial insemination.

Heterospermic AI is commonly used in swine despite preventing precise evaluation of individual boar fertility. The present study compared the contribution of four boars (A, B, C and D) for reproductive performance and for paternity using homospermic and heterospermic (AB, AC, AD, BC, BD and CD) AI (n=204 for homospermic AI; n=307 for heterospermic AI). Blood samples from the four boars, from all sows inseminated with heterospermic doses and from the umbilical cords of their piglets, as well as tissue smears from mummified fetuses, were genotyped using single nucleotide polymorphisms (SNPs). Differences among boars were detected for the in vitro oocyte penetration rate and for the number of spermatozoa per oocyte (P<0.05), but not for sperm motility, mitochondrial functionality and integrity of the membrane, acrosome and DNA (P>0.05). Homospermic and heterospermic AI resulted in similar (P>0.05) farrowing rates (90.5% and 89.9%, respectively) and total litter size (12.4±0.4 and 12.7±0.7, respectively). Farrowing rate was lower for Boar B than for Boar C (P<0.05), but no other differences in reproductive performance among boars were observed with homospermic AI. The SNPs determined the paternity of 94.2% of the piglets sired by heterospermic AI. In the AC pool, paternity contribution per boar was similar (P>0.05), but differences between boars occurred in all other pools (P<0.05). Boar D achieved the greatest paternity contribution in all pools and parity categories (nearly 60%), whereas Boar B sired the fewest piglets (at most 40%). Reproductive performance was similar with homospermic and heterospermic AI, but differences in performance among boars undetected with homospermic AI were only evident after genotyping the piglets sired through heterospermic AI.

EFFECT OF SOLID MEDIUM DURING COOLED STORAGE ON STALLION SPERM PARAMETERS.

BACKGROUND: Solid storage medium prevents cellular sedimentation, reduces metabolic demand via limiting movement, and avoids the modification of an extender composition in the sedimentary microenvironment. It has been proven to prolong spermatozoa viability in mammalians. OBJECTIVE: This experiment aims to evaluate the effect of cool storage in solid phase extender on stallion sperms. MATERIALS AND METHODS: Semen was collected from 10 Crioulo stallions (n=30) and submitted to treatments: control group (semen extender) and groups with gelatin addition in different concentrations (semen extender + 1%, 2% and 3%). Seminal analyses included motility, mitochondrial functionality, plasma membrane integrity, DNA and acrosome at 0; 24; 48 and 72 hours during cooled storage at 5 degree C. RESULTS: Motility, mitochondrial functionality, plasma membrane and acrosome integrity declined during storage time, with no statistical difference between treatments. DNA integrity did not significantly change during storage period. CONCLUSION: Solid medium was not harmful and did not improved stallion sperm parameters during cooled storage.

Sperm impairments in adult vesper mice (Calomys laucha) caused by in utero exposure to bisphenol A.

This study aimed to evaluate the effects of in utero administration of bisphenol A (BPA) on semen parameters of vesper mice. Sixty female Calomys laucha were divided into six groups and received by gavage during gestation the following substances: Water (negative control), Olive Oil (vehicle control), Diethylstilbestrol (DES - positive control - 6.5 µg kg(-1) bw) and BPA (40, 80 and 200 µg kg(-1) bw). Male offspring were euthanised at 70 days of age, and sperm parameters were analysed. BPA reduced normal sperm morphology (water = 96.1 ± 0.65; BPA200 = 96.8 ± 2.3%), sperm membrane integrity (water = 88.8 ± 1,65; BPA200 = 70.6 ± 4,15%), sperm motility (water = 87.5 ± 1.71; BPA200 = 51.3 ±9.9%) and in vitro penetration rates (water = 55.0 ± 7.14; BPA200 = 7.47 ±2.96%), but it did not affect body weight, anogenital distance, sperm DNA integrity and acrosome integrity. In conclusion, in utero exposure to BPA caused a reduction in sperm parameters of adult C. laucha. Natural mating studies should be conducted to verify the effects of BPA on fertility of the animals.

Influence of mineral, olive or sunflower oils on male reproductive parameters in vitro--the wild rodent Calomys laucha.

The aim of this study was to evaluate the influence of oils on male reproductive parameters in Calomys laucha. Twenty-four animals were distributed into four groups and given the following substances by gavage: water, mineral oil, olive oil and sunflower oil. After 10 days of gavage, the animals were euthanised and the semen was collected from them for assessing acrosome integrity and carrying out in vitro penetration (IVP) test. Acrosome was significantly reduced (P < 0.05) for the vehicles in relation to control. In vitro penetration was reduced in all vehicles in relation to control, but only sunflower oil had statistically lower levels of reduction (P < 0.05). Oily vehicles are able to influence in vitro reproductive tests negatively, interfering in reproductive toxicological studies.

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.

Protein profile as a quality indicator of cryopreserved semen from Tambaqui Colossoma macropomum (Cuvier, 1818).

The aim of this study was to evaluate the association between proteins in the seminal plasma of tambaqui Colossoma macropomum (Cuvier, 1818) with seminal quality indicators after thawing. The semen was cryopreserved with a dilution based on BTS with 8% DMSO. A 200 µL sample of semen from each animal was diluted in 800 µL BTS, centrifuged at 800 rpm, and the supernatant was cryopreserved to further analyze of the protein profile of seminal plasma through one-dimensional electrophoresis (SDS-PAGE). After 15 days of cryopreservation, a cryopreserved semen straw was thawed to analyze both qualitative and quantitative parameters. When considering all collections, the SDS-PAGE identified 15 protein bands in the seminal plasma of tambaqui. When the interaction (presence or absence) between proteins observed in the seminal plasma and the post thawed spermatic parameters was evaluated, we observed a great influence of the presence of proteins on spermatic quality. A greater (P<0.05) fertilization rate was observed with the presence of proteins 12, 34, 44, 85, and 90 kDa. Proteins in seminal plasma of tambaqui influenced the spermatic quality after thawing, and thus, they can be utilized as an indicator of sperm quality, especially the proteins with a molecular weight ≤ 50 kDa.