GONADOTROPIN AND SEX STEROID HORMONES IN MALES WITH POST COVID-19 INFECTION.

OBJECTIVE: The aim: To understand the effects of COVID-19 infection on gonadotropins and sex steroid hormones in males. PATIENTS AND METHODS: Materials and methods: This is a cohort study conducted in fifty males, who had been previously infected with COVID-19 with normal hormonal profile. Fifty Iraqi males were attending the male clinic at Higher Institute of Infertility Diagnosis and Assisted Reproductive Technologies, diagnosed with standard methods. The assessment of serum hormonal levels including (FSH, LH, Prolactin and Testosterone) was done 3 times: 1st time after one-month post recovery after COVID-19, 2nd time after 2 months post recovery and 3rd time - after 3 months post recovery. RESULTS: Results: There was no significant change in the mean level of serum FSH during the first, second and third months (p = 0.630). LH serum level was highly significantly reduced during follow up (p< 0.001). Serum prolactin level, reduced significantly during follow up (p< 0.001). Serum testosterone level was the lowest in the first month and increased during the second month and then during the third month in a highly significant manner (p< 0.001). CONCLUSION: Conclusions: Sub-clinical hypogonadism may be suspected as a consequence of COVID-19 infection in males as its first presentation characterized by increased LH & decreased testosterone production.

Extenders for alpaca epididymal spermatozoa: Comparison of INRA96 and andromed.

Artificial insemination would be a useful technique for alpaca breeders to use as an aid to breeding to increase fleece quality. The technique, however, is not well developed in alpacas, partly because of the viscous nature of their seminal plasma. Castration conducted for husbandry purposes can provide a source of epididymal spermatozoa to test semen extenders or handling regimens, thus circumventing the problem of the viscous ejaculate. In this experiment, two semen extenders (Andromed and INRA96) developed for other species (bovine and equine, respectively) were tested with alpaca spermatozoa derived from the cauda epididymis. Sperm total motility (mean ± SEM A: 29.1 ± 4.8 % compared with I: 35.4 ± 4.8 %; NS), membrane integrity (A: 58 ± 9% compared with I: 56 ± 9%; NS) and acrosome integrity (A: 65 ± 7% compared with I: 54 ± 7%; NS) were not different between the two extenders. Progressive motility with use of INRA96 was greater after incubating for 30 min than after incubating for 10 min (35 ± 4% vs. 12 ± 4%, respectively; P = 0.03). In conclusion, viable epididymal spermatozoa could be extracted from the castrated organs after overnight transport. There were no differences in sperm quality between the two extenders; therefore, it appears that either extender could be used for alpaca spermatozoa. These results could help in the development of a technique for artificial insemination in alpacas.

Season does not have a deleterious effect on proportions of stallion seminal plasma proteins.

The mechanism by which the content of the major groups of seminal plasma proteins in stallion semen changes between the breeding and non-breeding seasons remains unknown. Here, we investigated the proportions of non-heparin-binding, phosphorylcholine-binding, and heparin-binding proteins in seminal plasma with the aim of relating them to sperm quality and testosterone levels in good and bad freezer stallions. Only minor variations in the major protein groups were found between the breeding and non-breeding seasons. In the non-breeding season, a higher content of a subset of non-heparin binding proteins as well as of heparin-binding proteins was found. Analysis of semen characteristics revealed a somewhat contrasting picture. While only minor variations in sperm kinematics and sperm morphology were found between seasons, the flow-cytometric measurements of mitochondrial membrane potential and also, to some extent, reactive oxygen species production indicated lower sperm quality in the breeding season. Chromatin integrity and testosterone levels were unchanged between seasons. The results suggest that stallion ejaculates could be used year-round for freezing, since only minor differences in protein composition exist between the breeding and non-breeding seasons, as well as between good and bad freezers. In addition, sperm quality is not impaired during the non-breeding season.

Effect of adding heterologous versus homologous bovine seminal plasma prior to cryopreservation on bull sperm quality after thawing.

SummaryThe aim of this study was to investigate the effect of adding homologous or heterologous bovine seminal plasma (SP) to SP-free sperm samples before freezing on sperm quality after thawing. Ejaculates from bulls of known fertility were used as a source of SP. The SP was removed from further aliquots of the same ejaculates by colloid centrifugation to create SP-free sperm samples; the resuspended sperm pellets were treated with homologous or heterologous SP from high or low fertility bulls at 0%, 1% or 5% before freezing. After thawing, sperm quality was evaluated by computer-assisted sperm analysis and flow cytometry for membrane integrity, reactive oxygen species, chromatin structure, mitochondrial membrane potential and protein tyrosine phosphorylation. Data were analysed using Proc MIXED, SAS®. Post-hoc comparisons were adjusted for multiplicity using Tukey's method. The addition of SP resulted in significant differences in sperm quality, namely velocity class A, Velocity Straight Line (VSL), Velocity Average Path (VAP), Velocity Curved Line (VCL), Amplitude of Lateral Head Displacement (ALH), Hyperactive (HYP), reactive oxygen species (ROS) production and % DNA fragmentation index (DFI) (P<0.05 for each). Although adding 5% homologous SP from high fertility bulls was beneficial to sperm kinematics, 5% heterologous SP from high fertility bulls had a deleterious effect on chromatin integrity and on sperm velocity. In conclusion, adding SP may have either a beneficial effect or a deleterious effect depending on the individuals involved. It might be feasible to use this method to improve sperm quality in some circumstances.

Addition of seminal plasma to thawed stallion spermatozoa did not repair cryoinjuries.

Freezing and thawing processes induce structural and functional damage to sperm plasma membranes and internal organelles. Adding seminal plasma (SP) has been found to minimize or repair the cryoinjuries in some species. The objective of this study was to investigate whether adding SP from stallions of known freezability after thawing could repair cryoinjuries. Semen was collected from warmblood stallions (n = 8, three ejaculates/stallion) and processed by Single Layer Centrifugation (SLC) to remove SP prior to freezing. Pooled SP (5%) from bad freezer (BF) or good freezer (GF) stallions was added after thawing. Post-thaw sperm quality was assessed by flow cytometry in terms of chromatin integrity (%DFI), membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and MitoSOX. Sperm kinematics were also assessed by computer-assisted sperm analysis. The %DFI was lower in SLC control (C) than in BF or GF (P < 0.0001, P < 0.0003 respectively). The proportion of viable spermatozoa with intact cell membranes was higher in C than in SP treated groups (C vs. BF, P = 0.02; C vs GF, P = 0.05). There were fewer spermatozoa with low MMP and more with high MMP for C than GF (P = 0.006). The spermatozoa treated with SP from good freezers produced more ROS than when treated with SP from bad freezers (P = 0.007). Motility parameters were not affected by adding SP. In conclusion, adding SP after thawing does not have a beneficial effect on sperm quality, suggesting an inability to repair stallion sperm cryoinjuries, regardless of whether the SP originated from stallions semen, which has good or bad quality after thawing.

Seminal plasma influences the fertilizing potential of cryopreserved stallion sperm.

Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from "good" (GF) or "bad" (BF) freezer stallions on sperm cells' fertilizing ability. "Good freezers" refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst "bad freezer" stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the binding ability of equine sperm cells as a possible alternative to artificial insemination trials. The effect of adding SP i) prior to cryopreservation; ii) after thawing of sperm cells selected by single layer centrifugation (SLC); iii) to capacitation medium, was evaluated. Adding SP from GF stallions prior to cryopreservation reduced the mean number of sperm cells bound to the zona pellucida (ZP) compared to control (P = 0.0003), SP-free sperm cells and group received SP from BF stallions (P ≤ 0.0001 for both). After thawing SLC-selected sperm cells treated with 5% SP showed a decrease in binding ability compared with SP-free sperm cells (P ≤ 0.0001). The binding affinity of sperm cells was higher in the group treated with SP from GF than with SP from BF stallions (P ≤ 0.05). Prolonged exposure to SP impaired the ability of stallion sperm cells to undergo capacitation and bind to ZP, regardless of the source of SP (P ≤ 0.0001). The response of equine sperm cells to SP is influenced by the ability of the sperm cells to withstand cryopreservation and is affected by the timing of exposure and the origin of SP. Customization of the protocol for individual stallions is recommended to optimize the effect.

Adding bovine seminal plasma prior to freezing improves post-thaw bull sperm kinematics but decreases mitochondrial activity.

Variations in fertility between bulls with comparable sperm quality could be due to differences in their seminal plasma (SP). The aim of this study was to investigate the effect of adding bovine SP from bulls of known fertility to SP-free sperm samples. After removal of SP by Single Layer Centrifugation, resuspended sperm pellets were treated with SP from high or low fertility bulls at 0% (control), 1%, or 5% before freezing. Sperm quality was evaluated after thawing. Data were analyzed using Proc MIXED, SAS®. Bovine SP at 1% or 5% SP1 and SP5, respectively, decreased average path velocity, curvilinear velocity, and amplitude of lateral head displacement whereas wobble and linearity were increased. In addition, the proportion of spermatozoa with high mitochondrial membrane potential (MMP) was lowest for treatment with SP5 compared to SP1 and control. The proportion of SP did not affect other parameters of sperm quality. Thus, adding 5% bovine SP produced a favorable effect on some sperm velocity parameters but had an unfavorable effect on MMP. There were no differences in effect between SP from high and low fertility bulls. ABBREVIATIONS: AI: artificial insemination; BCF: beat cross frequency; CASA: computer-assisted sperm analysis; IVF: in vitro fertilization; MMP: mitochondrial membrane potential; SLC: single layer centrifugation; SP: seminal plasma.

Improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma.

Addition of seminal plasma (SP) prior to cryopreservation may influence stallion sperm cryosurvival. The objective of this study was to investigate the addition of pooled SP from "good" or "bad" freezer stallions to spermatozoa selected by single layer centrifugation (SLC) prior to cryopreservation on post-thaw sperm quality. Semen from 12 stallions was collected; 5 mL was frozen as control (C) and the remainder was processed by SLC to remove SP and was divided into three aliquots: i) SLC sample without SP (SLC); ii) SLC plus pooled SP from "good freezer" stallions (SLC-GF); iii) SLC plus pooled SP from "bad freezer" stallions (SLC-BF). After thawing, the following parameters were evaluated: chromatin integrity (DNA fragmentation index; %DFI), mitochondrial membrane potential (MMP), membrane integrity (MI), reactive oxygen species (ROS) and sperm kinematics. The %DFI was reduced (P < 0.0001) in SLC samples compared to controls. The SLC group showed a lower proportion of spermatozoa with low MMP and a higher proportion of spermatozoa with high MMP than other groups (P < 0.0001), and had lower hydrogen peroxide content than control. Sperm kinematics were not different. In conclusion, selection by SLC prior to cryopreservation improved post-thaw sperm quality; inclusion of SP from "good" and "bad" freezer stallions did not have an additional beneficial effect.

Sperm viability, reactive oxygen species, and DNA fragmentation index combined can discriminate between above- and below-average fertility bulls.

The accurate prediction of bull fertility is of major economic importance in the dairy breeding industry. Sperm fertilizing potential is determined by their ability to reach the oocyte, complete fertilization, and sustain embryogenesis, which is partly determined by the quality of sperm DNA. In the present study, we analyzed several sperm functions required for fertility, including DNA damage, in frozen-thawed spermatozoa of breeding bulls with different adjusted nonreturn rates (NRR56), and identified a suitable combination of parameters that could be used to predict bull fertility. Based on the NRR56, bulls were classified into below- and above-average fertility, a total of 37 characteristics of spermatozoa were evaluated for each bull, and their relationship with bull fertility was studied. Of the different sperm functional attributes, differences were observed in sperm viability, acrosomal integrity, reactive oxygen species, and DNA fragmentation index (%DFI) among below-average, average, and above-average fertility bulls. Principal component analysis also revealed that sperm viability, acrosome status, reactive oxygen species, and %DFI were the important variables, having highest correlation with NRR56. Our results indicated that the proportion of live [correlation coefficient (r) = 0.53] and live acrosome-reacted spermatozoa (r = 0.50) were significantly positively related to NRR56, whereas the proportion of dead spermatozoa (r = -0.53) and %DFI (r = 0.61) were significantly negatively related to NRR56 in bulls. Linear regression analysis indicated that a combination of live [coefficient of determination (R2) = 0.72], dead (R2 = 0.72), live hydrogen peroxide-negative spermatozoa (R2 = 0.64), and %DFI (R2 = 0.56) could differentiate below-average and above-average fertility bulls, and thus were considered for development of a fertility prediction model. The accuracy of the developed model for fertility prediction in bulls was high (R2 = 0.83). We concluded that flow cytometric detection of sperm viability, hydrogen peroxide status, and %DFI could discriminate below- from above-average fertility bulls.