Artificial Vagina Research Articles

Experience of preputiotomy performing in bull with acroposthitis and fibrous affect of penis’ S-shaped curve

The aim of this study was to perform a preputiotomy with the formation of an artificial opening in a breeding bull to realize his reproductive potential. Scientific and production research was conducted in the spring of 2024. In a 3.5-year-old Holstein breeding bull, kept at a breeding station for keeping breeding bulls, acroposthitis and fibrous lesion of the penis in the area of the S-shaped bend were detected due to the developed infectious process caused by Pseudomonas aeruginosa. The resulting pathology prevented the penis from fully extending beyond the preputial sac when collecting sperm for an artificial vagina (the last sperm collection was performed on 02/07/2024). The operation performed consisted of the following stages: insertion of a thick-walled tube into the preputial sac for better orientation in the tissues; making a V-shaped incision of all layers of the prepuce near its fornix; excision of the prepuce section necessary for free removal of the penis when collecting sperm; formation of an artificial opening by connecting the parietal preputial layer to the skin with an interrupted suture. The postoperative period included daily monitoring of the animal's condition, antibiotic therapy (amoxicillin (0.15 g), three injections every 48 hours), and the use of non-steroidal anti-inflammatory drugs (meloxicam (0.002 g), 3 days). A month after the operation, sperm was collected (06.05.2024, three months later) and the presence of all unconditioned reflexes was noted. At the same time, the copulatory reflex was full, painless and ended in ejaculation. The obtained volume of sperm after two mountings was 2.5 ml, the concentration was 1.3x108, the activity was 8 points. Thus, preputiotomy is an effective method for surgical correction of lesions of the preputial sac and adhesions in the area of the S-shaped bend of the penis in breeding bulls.

GROWTH PERFORMANCE AND SEMEN CHARACTERISTICS OF MONGREL RABBIT BUCKS FED DIETARY LEVELS OF GARLIC (ALLUM SATIVUM) MEAL

This study was designed to evaluate the effect of garlic meal supplementation on growth performance and semen characteristics in rabbit bucks. Twenty-four (24) mixed breed rabbit bucks were used for this study. The bucks were allowed to acclimatize for two weeks before the commencement of the study, during this period, they were fed formulated diet and Calopogonium ad-libitum. Feed and clean drinking water were offered ad libitum to the experimental animals. Four different experimental diets were formulated with varying levels of 0.00, 0.50, 1.00 and 1.50% garlic meal as supplement and were coded as T1, T2, T3 and T4 respectively, with as control. The 24 bucks were weighed and randomly allotted into the four experimental treatments and replicated thrice with two rabbit bucks per replicate in a complete randomized design (CRD) for a period of 8 weeks. At the end of the feeding period, semen collection was done using a specially designed artificial vagina for rabbits. The data obtained were subjected to one-way analysis of variance (ANOVA) procedure using IBM Statistical Package for Social Science (SPSS) version 21. The result showed that garlic meal had significant effect (P<0.05) on final weight, total and daily feed intake. Garlic significantly influenced (p<0.05) sperm volume, motility, concentration and total cells per ejaculate however, did not affect sperm morphology. In conclusion, garlic meal supplementation at 1.0% (10g/kg) can improve some growth and seminal parameters in male rabbits.

Effect of Skim Nanoparticles on the Motility and Kinematics of Simmental Cattle Frozen Semen

Nanotechnology has positively impacted the development of extenders used in the cryopreservation of spermatozoa in livestock. The inclusion of nano skim in the extender is expected to preserve semen quality after thawing. This study aimed to determine the optimal concentration of nano skim. Various concentrations of nano skim (0%, 1.66%, 3.33%, 5%, 6.66%, 8.33%, and 10%) with the addition of 10% nano egg yolk in each extender were compared. Fresh semen samples were collected from three three-year-old Simmental bulls using an artificial vagina. The study parameters included motility (total motility, progressive motility) and kinematics (velocity curve linear (VCL), velocity straight linear (VSL), velocity average pathway (VAP), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and wobble (WOB). A completely randomized design was employed, and data were analyzed using analysis of variance (ANOVA). Fresh semen dilution results indicated that only the control group (0%) and the nano skim group with concentrations of 6.66% and 8.33% were eligible for further processing into frozen semen. The results showed no significant difference between the three treatment groups on sperm motility and kinematics of Simmental cattle after thawing (p0.05). The control group exhibited the highest percentage of total motility, VCL, VAP, and ALH values, while the 6.66% nano skim group outperformed the 8.33% nano skim group. The highest percentage of progressive motility and VSL value was observed in the 6.66% nano-skim group compared to the 8.33% nano-skim and control groups. The highest values of BCF, WOB, LIN, and STR was observed in the nano-skim group at 8.33% compared to the nano-skim group at 6.66% and control. The study concluded that 6.66% nano skim was the optimal concentration to maintain the quality of frozen semen of Simmental cattle.

Effects of Time Storage, Egg Yolk Concentration and Semen Washing on Sperm Motility in Arbia Bucks

Abstract With a prospect to improve goat artificial insemination, this study aims to evaluate the effects of storage time, egg yolk concentration and semen washing on the rate of motile spermatozoa (spz) in Arbia breed bucks semen stored at 5 °C for 48 h. Semen was collected from three mature bucks using an artificial vagina during December 2018. The ejaculates used in this study (rate of motile sperm > 75 %) were divided into two fractions: A, and B, (unwashed and washed samples, respectively): themselves divided into three fractions, and diluted in Tris-Acid citric based extenders, containing egg yolk at 2 %, 10 % and 20 %. ANOVA test was performed using the R software version 3.3.0. The rate of motile spermatozoa was evaluated after 24 and 48 hours of storage in the refrigerator at 5 °C. It registered a significant decrease as a function of the storage time in the different extenders. It varied between 42.43 ± 9.86 % and 61.29 ± 5.44 %, after 24 hours of storage, and between 35.86 ± 13.95 %, and 55.45 ± 11.65 % after 48 hours of storage. The semen washing significantly reduced the rate of motile spermatozoa in the different extenders. Increasing the concentration of egg yolk in the extender increased the rate of motile spermatozoa, especially during the first 24 hours of storage. In conclusion, the rate of spermatozoa motility of unwashed semen stored at 5 °C is higher in extenders containing high egg-yolk concentration, and decrease according to conservation time.

Effect of cooling rates and equilibration times on post-thaw sperm quality of Kail rams

Context The conflicting findings regarding the impact of equilibration time on post-thawed sperm quality underscore the need for further research to evaluate the impact of equilibration time and cooling rate on post-thaw sperm quality of ram semen. Aims The current study aimed to assess the combined impact of cooling rates and pre-freezing equilibration times on post-thaw sperm quality in Kail ram semen (n = 5). Methods Semen collection was performed using an artificial vagina at 42°C. The pooled semen was divided into equal aliquots and subjected to either slow cooling (SC, −0.27°C/min) or moderate cooling (MC, −0.36°C/min) rates, transitioning from 37°C to 4°C. Equilibration times of 0, 4, 8, and 12 h were employed before freezing. Key results Semen samples undergoing the SC rate and equilibrated for 4 h exhibited higher (P < 0.05) percentages of progressive motile (PM), rapid progressive (RP), and medium progressive (MP) sperm compared with the MC rate. However, total motility remained unaffected by the cooling rate (P < 0.05). Semen equilibrated for 4 h demonstrated higher (P < 0.05) percentages of PM and RP sperm, as well as improved kinematics (curvilinear velocity, average path velocity, and straight-line velocity) compared with other equilibration times. Nevertheless, equilibration time had no (P > 0.05) impact on the amplitude of the lateral head displacement for semen samples subjected to the MC rate. Notably, the cooling rate did not affect post-thaw sperm kinematics, plasma membrane integrity, or live-sperm percentage (P > 0.05). Semen samples equilibrated for 4, 8, and 12 h exhibited a higher (P < 0.05) percentage of sperm with intact plasma membrane and viability than did those equilibrated for 0 h. Conclusions In conclusion, slow cooling rate and a 4 h equilibration period were shown to be optimal for preserving post-thaw sperm quality in Kail rams. Implications The findings highlighted the combined effect of cooling rate and equilibration time on post-thaw sperm quality for optimising sperm cryopreservation protocols in the context of ram semen.

The influence of rutin and chlorogenic acid on oxidative stress and in vivo fertility: Evaluation of the quality and antioxidant status of post-thaw semen from Azari water buffalo bulls.

The vulnerability of buffalo sperm to cryoinjury necessitates the improvement of sperm cryo-resistance as a critical strategy for the widespread use of assisted reproductive technologies in buffalo. The main aim of the present study was to evaluate the effects of different concentrations of rutin and chlorogenic acid (CGA) on buffalo semen quality, antioxidant activity and fertility during cryopreservation. The semen was collected and pooled from the 3 buffaloes using an artificial vagina (18 ejaculations). The pooled sperm were divided into nine different groups: control (Tris-based extender); 0.4, 0.6, 0.8 and 1mM rutin (rutin+Tris-based extender); and 50, 100, 150 and 200µM CGG (CGA+Tris-based extender). Sperm kinematics, viability, hypo-osmotic swelling test, mitochondrial activity, antioxidant activities and malondialdehyde (MDA) concentration of frozen and thawed buffalo sperm were evaluated. In addition, 48 buffalo were finally inseminated, and pregnancy was rectally determined 1 month after insemination. Compared to the control group, adding R-0.4, R-0.6, CGA-100 and CGA-150 can improve total and progressive motility, motility characteristics, viability, PMF and DNA damage in buffalo sperm. In addition, the results showed that R-0.4, R-0.6, CGA-50, CGA-100 and CGA-150 increased total antioxidant capacity, catalase, glutathione peroxidase and glutathione activities and decreased MDA levels compared to the control group. Furthermore, it has been shown that adding 150µM CGA and 0.6mM rutin to an extender can increase in vivo fertility compared to the control group. In conclusion, adding rutin and CGA to the extender improves membrane stability and in vivo fertility of buffalo sperm by reducing oxidative stress.

The effect of supplementing freezing extender with Mn2+-, Zn2+- or Cu2+-nanosuccinate on select post-thaw characteristics of ram semen

The effects of Mn2+-, Zn2+- or Cu2+-nanosuccinate added to freezing extender on select post-thaw semen characteristics were determined in six Texel rams (aged 2–4 years) during seasonal anestrus (April-May). Ejaculates (n = 6 per ram) collected into an artificial vagina were divided into ten isovolumetric fractions each. Semen was diluted in lactose-yolk-tris-citrate-glycerin medium and nanosuccinates (Mn2+- and Zn2+-nanosuccinate: 0.0 (control), 2.5, 5.0 and 7.5 μg/l; Cu2+-nanosuccinate: 0.0 (control), 1.25, 2.5 and 3.75 μg/l) were added to semen extender. Extended semen was loaded into 0.25-ml straws and frozen in liquid nitrogen. After thawing, sperm motility parameters were determined with computer assisted semen analysis (CASA), and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) was measured with a spectrophotometric technique. The addition of 5.0 μg/l of Mn2+- and Zn2+-nanosuccinate significantly increased the sperm progressive motility and both 2.5 and 5.0 μg/l improved sperm motion kinetics. Further, both nanosuccinates at a dose of 5.0 μg/l significantly decreased SOD activity and stimulated an increase in GPx and CAT activity in semen samples. Alternatively, the addition of Cu2+-nanosuccinate (highest dose) significantly reduced the progressive motility and velocity of ram spermatozoa, increased the percentage of sperm with acrosomal/head defects and seminal SOD activity, and depressed CAT (highest dose) and GPx (all doses) activity. In summary, the addition of Mn2+- and Zn2+-nanosuccinate to semen extender had beneficial effects on sperm motility/motion kinetics and structural integrity, whereas Cu2+-nanosuccinate generally had debilitating effects on the post-thaw semen characteristics in rams.

Repeated injections of vitamin E and Se improves testicular morphology, testosterone and in vitro and in vivo sperm fertility in subfertile rabbits

Subfertility is a multifactorial disorder that affects the rabbit production industry. However, subfertility may be treated by using a simple intervention such as vitamin supplementation. Vitamin E and selenium (Se) are potent antioxidants that protect the male reproductive system. The aim of this study is to determine the effects of vitamin E and Se on testicular size, semen quality and freezability, antioxidant activity, testosterone levels, and fertility in subfertile rabbits. Twenty-one New Zealand rabbits were classified as subfertile rabbits based on their semen characteristics and fertility records. The rabbits were randomly allocated into 3 equal groups (G1: control; G2: injected with Vit E 100 IU/head + Se 0.1 mg/kg b.w.; G3: injected with Vit E 200 IU/head + Se 0.2 mg/kg b.w. once weekly for 8 weeks).Once weekly for 8 W, blood samples were collected to measure serum testosterone level and total antioxidant capacity (TAC), and semen samples were collected by artificial vagina to assess the quality of fresh and frozen semen. At the 8th week of the study, 150 multiparous does were artificially inseminated with fresh semen to assess the fertility of rabbits after treatment; 50 does for each group. At the end of the study, rabbits were slaughtered to assess testicular morphometry. Fresh and post-thaw semen quality parameters were significantly (p < 0.05) higher in G3in comparison with G2and G1, respectively. Also, testosterone level was significantly (p < 0.05) increased at the 2nd week in G3in comparison with other groups. Conception and kindling rates were significantly (p < 0.05) higher in does which were inseminated with semen fromG3. In conclusion, injection of vitamin E and selenium at a higher dose (G3) improved the testicular morphology, quality of fresh and post-thaw semen, and most importantly, the fertility of subfertile rabbits.

Semen microorganisms’ prevalence in tabapuã bulls

The study objective was to characterize the microbiological aspects of raw semen from Tabapuã bulls kept in a semen production center. Five Tabapuã bulls were used, and four ejaculates were collected using the artificial vagina method. Bacterial colonies were quantified and identified by morphotintorial analysis (Gram staining) and identification tests for bacterial isolates. Among the twenty semen samples collected, bacteria genera were identified: Citrobacter sp. (3.03%); Corynebacterium sp. (3.03%); Enterobacter sp. (9.09%); Escherichia sp. (9.09%); Klebsiella sp. (9.09%); Providencia sp. (3.03%); Pseudomonas sp. (12.12%); Staphylococcus sp. (45.46%) and Streptococcus sp. (6.06%). It was possible to observe and identify the microbial agents present in the semen samples.

Seasonal variation of NGF in seminal plasma and expression of NGF and its cognate receptors NTRK1 and p75NTR in the sex organs of rams

Nerve growth factor (NGF) has long been known as the main ovulation-inducing factor in induced ovulation species, however, recent studies suggested the NGF role also in those with spontaneous ovulation. The first aim of this study was to evaluate the presence and gene expression of NGF and its cognate receptors, high-affinity neurotrophic tyrosine kinase 1 receptor (NTRK1) and low-affinity p75 nerve growth factor receptor (p75NTR), in the ram genital tract. Moreover, the annual trend of NGF seminal plasma values was investigated to evaluate the possible relationship between the NGF production variations and the ram reproductive seasonality. The presence and expression of the NGF/receptors system was evaluated in the testis, epididymis, vas deferens ampullae, seminal vesicles, prostate, and bulbourethral glands through immunohistochemistry and real-time PCR (qPCR), respectively. Genital tract samples were collected from 5 adult rams, regularly slaughtered at a local abattoir. Semen was collected during the whole year weekly, from 5 different adult rams, reared in a breeding facility, with an artificial vagina. NGF seminal plasma values were assessed through the ELISA method. NGF, NTRK1 and p75NTR immunoreactivity was detected in all male organs examined. NGF-positive immunostaining was observed in the spermatozoa of the germinal epithelium, in the epididymis and the cells of the secretory epithelium of annexed glands, NTRK1 receptor showed a localization pattern like that of NGF, whereas p75NTR immunopositivity was localized in the nerve fibers and ganglia. NGF gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis than in the other tissues. NTRK1 gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.05) in all the other tissues examined. Gene expression of p75NTR was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis and bulbourethral glands. NGF seminal plasma concentration was greater from January to May (p < 0.01) than in the other months. This study highlighted that the NGF system was expressed in the tissues of all the different genital tracts examined, confirming the role of NGF in ram reproduction. Sheep are short-day breeders, with an anestrus that corresponds to the highest seminal plasma NGF levels, thus suggesting the intriguing idea that this factor could participate in an inhibitory mechanism of male reproductive activity, activated during the female anestrus.

Influence of incorporating L-carnitine or Moringa oleifera leaves extract into semen diluent on cryosurvival and in vitro fertilization competence of buck sperm

This study aimed at scrutinizing efficiency of incorporating L-carnitine or M. oleifera leaves extract into semen diluent on improving cryopreservation capacity and in vitro fertilization ability of buck spermatozoa. Ejaculates (n=48) were collected by an artificial vagina from six adult Damascus bucks twice weekly during the breeding season (September–October). Following initial evaluation, ejaculates of each collection session from the same bucks were pooled, diluted (1:10) with glycerolized (3 % glycerol, v/v) tris-citric acid egg yolk diluent and were split into three aliquots. The first aliquot served as control, whereas the second and third aliquots were supplemented with 4 μL/mL L-carnitine and 400 μL/mL moringa leaves extract (v/v), respectively. Thereafter, all specimens were processed for cryopreservation and were stored in liquid nitrogen (-196 °C) for 12 months before post-thaw sperm criteria were analyzed by a computer-assisted sperm analysis (CASA) system. Integrity of sperm DNA post thawing was visualized in all semen groups by fluorescence imaging, and in vitro fertilization ability of spermatozoa was also determined. Inclusion of L-carnitine or moringa leaves extract into the diluent improved (P<0.05) post-thaw sperm physical, morphofunctional and kinematic attributes, whilst maintaining (P<0.05) integrity of sperm DNA throughout the freezing and thawing cycle. Consequently, both supplemented groups yielded higher (P<0.05) in vitro fertilization rates compared to control. These results accentuate the protective roles of these antioxidants on buck sperm against consequences of cryopreservation-induced oxidative stress, hence ameliorating post-thaw sperm quality and fertilization competence. This is crucial for successful application of AI and IVF in goat selective breeding programs.

First dose optimization study on freezing Anatolian buffalo semen

The main objective of sperm production centers is to produce as many straws as possible from the obtained ejaculates using the optimal dilution rate. To this end, this study is the first to evaluate the effect of different semen extender rates on Anatolian buffalo semen quality. Ejaculates were collected by artificial vagina from three Anatolian buffalo bulls. These ejaculates were divided into three aliquots and filled into 0.25 ml straws with soy-based extenders at concentrations of 35, 25, and 15 million sperm/straw (n=105). The straw samples of different sperm concentrations were frozen. The quality of sperm was evaluated after thawing (37 °C, 30 sec) and following the thermoresistance test (37 °C, 3 h). The post-thaw total motility and progressive motility values were similar between the groups. However, following the thermoresistance test, there was a significant decrease in total motility in the 35 million sperm/straw group, and the progressive motility was significantly higher in the 25 million sperm/straw group. There was no statistically significant difference between the groups in terms of sperm kinetic parameters, except for VSL after thawing, as well as VAP and LIN values following the thermoresistance test. The overall mean PMAI and STR values were the highest in the 25 million sperm/straw group. In conclusion, it is recommended to dilute the Anatolian buffalo semen at a concentration of 25 million/0.25 ml when freezing it with a soy-based semen extender. In addition, it is considered that soy-based extenders compensate for cryo-damage to sperm motility for a short time, and the thermoresistance test should be applied for objective evaluation in dose optimization studies.

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

Subtle membrane changes in cryopreserved bull spermatozoa when modified temperature drop rates are used during the first phase of freezing

BACKGROUND: Cryopreservation of spermatozoa involves reduction of temperature to a subzero level, leading to increased longevity. However, temperature reduction has a significant effect on sperm membranes. OBJECTIVE: To evaluate the impact of the rate of temperature drop during the first phase of freezing on subtle membrane changes in cryopreserved bull spermatozoa. MATERIALS AND METHODS: Thirty-two ejaculates from four bulls (eight ejaculates/bull) were collected using artificial vagina while keeping a 3 to 4 days gap between two collections. Diluted semen samples were equilibrated at 5°C for 4 hours. The samples were then placed in a pre-programmed semen freezer. The first phase of freezing, that is, 5°C till −10°C was subjected to three different temperature drop rates: accelerated (F1), moderate (F2), and slow (F3), at 20°C per min, 10°C per min and 5°C per min, respectively. After thawing, spermatozoa were assessed for percentage live, plasma, and acrosomal membrane integrity, along with the external appearance of phosphatidyl serine, indicating apoptosis. RESULTS: A significant difference (p&lt;0.05) in viability, plasma membrane integrity (HOS test), and acrosome membrane integrity (PSA test) was observed between F3 and the other groups. However, the parameters did not significantly differ between F1 and F2. The annexin V-PI assay (AN/PI) categorized four types of sperm populations: non-apoptotic and viable (AN−/PI−), apoptotic and viable (AN+/PI−), non-apoptotic and non-viable (AN−/PI+), and apoptotic and non-viable (AN+/PI+). The proportion of spermatozoa with (AN−/PI−) and (AN+/PI+) differed significantly (p&lt;0.05) between F3 and the other groups. The values for apoptotic and viable (AN+/PI−) and non-apoptotic and non-viable (AN−/PI+) sperm were not significantly different among all freezing categories. CONCLUSION: A slower temperature drop rate (freezing rate) during the first phase of freezing results in less damaging, subtle membrane changes.

Dietary supplementing South African indigenous rams with flaxseed oil and ascorbic acid improves cryopreserved semen quality and in vitro fertility

The purpose of this study was to evaluate how ascorbic acid with dietary flaxseed oil affects the quality and fertility of cryopreserved ram sperm in South African indigenous rams. Treatment diets were supplemented 60 days before semen collection to afford proper spermatogenesis, adaptation to the feed formulated and fed throughout the study. Semen was collected with the use of artificial vagina following dietary supplementation with five treatment diets (neg. cont. – negative control, pos. cont. – positive control, FLO – 5% Flaxseed oil, ASA – 4% Ascorbic acid, and FLO + ASA). Semen was then extended using tris-based extender and cryopreserved using the programmable freezer (CBS Freezer 2100 series, Laboratory consumables & chemical suppliers, America). Ovaries were collected from a neighbouring slaughter house and conveyed to the lab in 0.9% saline at 37 °C. Data (sperm parameters and in vitro fertility) was then exposed to the GLM (General Linear Model) in Minitab 17. Pearson’s correlation coefficient was utilized to investigate the relationship between cryopreserved sperm quality and in vitro fertility. The student Least Significant Difference Test was used to separate the treatment means, and differences were accepted when the p-value was less than 0.05. The FLO + ASA group had higher (p < 0.05) progressive (36.33 ± 1.87), total (88.24 ± 2.24), rapid motility (27.52 ± 1.74), intact plasma membrane (75.67 ± 2.08), total fertilization (65.98 ± 7.39), and total cleavage (66.19 ± 6.50) when compared to other treatment groups. Total fertilization rate had a medium significant (p < 0.001) medium correlation with the progressive motility (r2 = 0.435), total motility (r2 = 0.447) and rapid motility (r2 = 0.409). In conclusion, dietary flaxseed and ascorbic acid (FLO + ASA) improves cryopreserved semen quality, in vitro fertilization rate, and the total cleavage rate. Noteworthy, the progressive, total and rapid motility play a crucial in the in vitro fertilization rate.

Potential use of soy lecithin or butylated hydroxytoluene as an alternative to powdered egg yolk for ram semen cryopreservation

The aim of this study was to assess the effect of replacing powdered egg yolk (PEY) with soybean lecithin (SL) or butylated hydroxytoluene (BHT) on ram sperm cryopreservation. Two ejaculates/male were collected via artificial vagina from 8 rams during breeding season. Ejaculates were pooled and washed twice by centrifugation. The pellet was divided into three aliquots, diluted in a Tris-based media with 5% glycerol containing PEY (15%), SL (1%) or BHT (0.6 mM) and cooled for 4 h at 5 °C before freezing. Sperm motility, plasma and acrosome membrane integrity and mitochondria activity as lipid peroxidation were assessed immediately after thawing and after 4 h of resilience incubation in a modified PBS at 38 ºC. After thawing, sperm extended in BHT showed the poorest quality compared to sperm extended in PEY and SL. Similar total and progressive motility were observed in sperm preserved in PEY and SL media. Plasma membrane integrity, however, was significantly higher in sperm extended in SL, although most of them with non-functional mitochondria. Acrosome damage was significant lower in SL sperm samples compared to PEY samples. Highest level of lipid peroxidation was found in sperm preserved in PEY. Resilience test had a negative effect (P &lt; 0.05) on plasma and acrosome membrane integrity in all samples, and on progressive motility only in sperm preserved in PEY. In conclusion, soy lecithin could be a potential alternative to PEY for ram cryopreservation, although its adverse effect on sperm mitochondria function has to be strongly considered.

Various Saccharides Addition of Extender Ram Efficiency to Improve Cryopreservation in Semen Etawah Crossbreed Bucks with Seminal Plasma Replacement

Phospholipase in buck seminal plasma will trigger a coagulation incident resulting in the demise of the spermatozoa during liquid semen processing using milk or egg yolk substrates diluent. A plasma replacement process is performed to avoid this possibility. The cryopreservation process leads to sperm cell damage due to the freezing process, therefore cryoprotectant agents such as saccharides are required as protective agents. This study aimed to investigate the effect of various types of saccharides on the quality of frozen semen of etawah crossbreed (PE) bucks during cryopreservation and thawing with plasma replacement using Priangan ram semen plasma. Semen was collected using an artificial vagina once a week. Fresh PE bucks semen centrifuged at 3,000 RPM for 30 min. The supernatant (seminal plasma) was removed and replaced in equal volume with Priangan ram seminal plasma. Semen was divided into four treatments: goat semen with sheep semen plasma in Tris diluent (control); control with 0.6% dextrose added (monosaccharides); control with 0.6% sucrose added (disaccharides); control with 0.6% raffinose added (trisaccharides). Semen quality including percentages of motile spermatozoa, live spermatozoa, and intact plasma membrane (IPM) were evaluated after diluted, equilibrated, and thawing, respectively. Results of this study showed that viability and IPM of monosaccharides, disaccharides, and trisaccharides were significantly (P&lt;0.05) higher than control (54.40 and 51.40; 55.00 and 53.60; 55.60 and 52.20 vs 48.40 and 52.20, respectively). The motility of disaccharides was significantly (P&lt;0.05) higher compared to control and other diluent (47.00 vs 41.00, respectively). In conclusion, different types of saccharides were effective in maintaining the quality of etawah crossbreed buck frozen semen. The addition of disaccharides seems more effective compared to monosaccharides and trisaccharides in tris extender on the quality of etawah crossbreed buck frozen semen.

Supplementation with MitoTEMPO before cryopreservation improves sperm quality and fertility potential of Piedmontese beef bull semen.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 μM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 μM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 μM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 μM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 μM and the mitochondrial membrane potentials was increased at 80 μM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 μM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 μM as an additive in the cryopreservation media of bull semen to improve sperm quality.

An Overview of Artificial Insemination: A Journey from Past to Present

Artificial insemination (AI) has a rich history spanning centuries, beginning with early experiments in plant and animal reproduction. This article traces the development of AI from its earliest recorded instances in 1322 A.D. to modern-day advancements. Notable pioneers such as Spallanzani, Hunter, and Ivanoff contributed significantly to the evolution of AI techniques. The introduction of AI across various animal species, including cattle, horses, sheep, goats, pigs, and poultry, revolutionized breeding practices worldwide. Key milestones include the development of semen extenders, the invention of artificial vaginas for semen collection, and the advent of cryopreservation techniques. Moreover, advancements in sperm quality assessment and estrous synchronization further enhanced AI efficiency. The introduction of sexed semen technology has provided a means to control offspring gender, offering new avenues for genetic selection. Overall, AI has played a crucial role in improving livestock genetics, disease control, and reproductive efficiency, making significant contributions to agricultural productivity and sustainability.

Oral supplementation with Scabiosa artropurperea L. aqueous extract improves the invivo sexual behaviour, sperm quality and androgen level in rams.

The effects of an aqueous extract of Scabiosa atropurpurea L. (AES) on the reproduction potential of Queue Fine de l'Ouest rams were evaluated over 9 weeks. Eighteen mature (4-6 years old) rams (52.8 ± 2.6 kg) were divided into three groups. The control (C) group was fed oat hay adlibitum with 700 g of concentrate and the other two groups were fed the same diet supplemented with AES at 1 and 2 mg/kg body weight (AES1 and AES2, respectively). Ram sperm was collected with an artificial vagina (2 × 2 days/week) to evaluate sperm production and quality, antioxidant activity, the adenosine triphosphate (ATP) and calcium concentrations. Sexual behaviour and plasma testosterone concentrations were also investigated. The administration of AES improved sexual behaviour (the duration of contact and the number of lateral approaches). The addition of AES also improved individual spermatozoa motility (C: 71.7% ± 6.3%; AES1: 78.3% ± 4.9%; AES2: 83.8% ± 4.4%), the sperm concentration (C: 5.6 ± 0.36; AES1: 6.4 ± 0.81; AES2: 6.7 ± 0.52 × 109 spermatozoa/mL), the ATP ratio (C: 1 ± 0.08; AES1: 2.1 ± 0.08; AES2: 3.3 ± 0.08) and the calcium concentration (C: 5.6 ± 0.24; AES1: 7.7 ± 0.21; AES2: 8.1 ± 0.24 mmol/L). AES treatment decreased the percentage of abnormal sperm (C: 18.5% ± 1.2%; AES1: 16.2% ± 1.1%; AES2: 14.8% ± 0.94%) and DNA damage (C: 62%; AES1: 27%; AES2: 33%) and was associated with elevated seminal fluid antioxidant activity (C: 22 ± 0.27; AES1: 27.1 ± 1.08 and AES2: 27.5 ± 0.36 mmol Trolox equivalents/L) and plasma testosterone (C: 8.3 ± 0.7; AES1: 11.7 ± 0.4; AES2: 15 ± 0.7 ng/L). In conclusion, our study suggests that S. atropurpurea may be potentially useful to enhance libido and sperm production and quality in ram.