PSXII-17 Fertility prediction model for Nilli-Ravi buffalo bulls through Fourier harmonic analysis of sperm

A model to predict Nili-ravi buffalo bull fertility was developed based on Fourier harmonic analysis of sperm. Seventeen bulls with 3032 AI records were categorizes based on fertility rate (FR) as low (36.5±0.2, n = 6: SD< ˗1 from mean FR), medium (39.9±0.2, n = 3; SD +1 to -1 from mean FR) and high fertility (41.4±0.1, n = 8; SD > +1 from mean FR). Cryopreserved semen samples from these bulls were investigated for Fourier harmonic analysis of sperm nuclear shape. Hoechst-33342 and YOYO-1 fluorescent stains were used to identify live and dead sperm. Digital images were analyzed to get sperm nuclear perimeter points at different phase angles to generate Fourier functions. Mean harmonic amplitude (HA) 0 was different (P < 0.05) for 1700 live vs. 1294 dead sperm from the 17 bulls, thus live sperm were used for remaining analyses. The mean, variance, skewness and kurtosis values of 100 live sperm nuclei/bull were compared for HA0-5 between high (n = 6) and low (n = 6) fertility groups, considering equal number of bulls in each category. The mean HA2 (0.739±0.01 vs 0.686±0.00) and 4 (0.105±0.001 vs 0.007±0.001) were higher in high vs low fertility group respectively (P < 0.05). Sperm nuclear perimeter among high fertility group sperm was more elongated. There was also an increased skewness of HA0 as fertility increased (P < 0.05). Discriminant analysis defined a fertility model by using mean HA4, skewness HA0 and variance HA2, that resulted in 91.7% bulls into their correct fertility group upon cross-validation (canonical correlation=0.928; P < 0.05). Higher values of mean HA4, skewness HA0 and variance HA2 increase the chance of bulls being placed in the high fertility group. In conclusion, sperm nuclear shape in Nili-ravi buffalo bull is related to in vivo fertility. A fertility model using reported discriminant measures could be used to objectively identify Nili-ravi buffalo bulls of varying fertility.

L-Carnitine Improves Cytoprotection during Cryopreservation: A case study on Nili-Ravi Buffalo Sperm

The current study was aimed to evaluate the antioxidative effect of L-Carnitine at post thawing following cryopreservation of Nili-Ravi buffalo sperm. For this purpose, semen from three buffalo bulls were collected for 3 weeks using an artificial vagina (N=18; replicates). The qualified ejaculates were diluted employing tris-citric acid extender i.e., control did not receive any L-Carnitine and experimental groups having 0.5, 1.0, and 1.5 ng/mL of L-carnitine at 37 C with approximately 50 x 106 sperm/mL. The semen was cooled at 4 C and then equilibrated (4 hours), filled in straws (0.5 mL) at4 C, placed on LN2 vapours for 10 min, and kept into an LN2 container. The thawed semen was evaluated for post-thaw quality. The integrity of the sperm plasma membrane and motility (P?0.05) was highest in the extenders having 1.0 ng/mL of L-carnitine as compared to the control(received no L-Carnitine). However, sperm chromatin integrity and viability(live sperm with intact acrosome) remained similar. It was concluded that supplementing 1.0 ng/mL L-Carnitine of extender can improve the post-thaw quality of cryopreserved sperm. Based on the results of the current experiments it is recommended to include L-carnitine extender to improve post-thaw quality of buffalo sperm in terms of its motility and integrity ofits plasma membrane. Keywords: Buffalo, Sperm, Cryopreservation, Extender, L-Carnitine, Artificial insemination.

​Association of Bio-acoustic Features of Vocal Signals with Age and Semen Quality in Sahiwal Bulls

Background: Bio-acoustic features of animal’s voice can provide meaningful information about their biological and physical characteristics. The present study was conducted to get indicators of age from voice analysis and explore the relationship between voice features and seminal parameters in Sahiwal bulls. Methods: Voice samples were collected from healthy bulls (n=20), maintained at ICAR-NDRI, Karnal. Bulls were classified into two groups i.e. young bulls and adult bulls. The voice signals were analyzed by Adobe Premium software and acoustic features were extracted by using PRAAT software. Result: The mean of acoustic features viz. call duration (sec), mean intensity (dB), total energy (P2S), amplitude (P), pitch (Hz), unvoiced frame (%), jitter (%), bandwidth (Hz) mean N/H ratio (%) have been found significantly different while mean H/N ratio (dB), shimmer (%) and pulses were not found statistically (P greater than 0.05) different between adult and young bulls. The seminal parameters viz mass activity (0-5 scale), individual progressive motility (%), live sperm count (%) and total sperm abnormality (%) were found significantly different between adult and young bulls. No significant association between voice features and semen quality of bulls was observed. Hence, voice signals of male might provide some clues about their age but for semen quality, there is further need to explore the interesting relationship between voice features and fertility of breeding bulls.

Karakteristik dan kualitas semen sapi Sumba Ongole dalam pengencer BTS yang dimodifikasi dengan susu kedelai

This study aimed to find out the quality of fresh semen from Sumba Ongole (SO) bulls which was diluted with Bestvile Thawing Solution (BTS) diluent modified with soybean milk (SKD). The study used a completely randomized design consisting of four treatments and 5 replications, P0 (100 % BTS), P1 (95 % BTS + 5 % SM), P2 (90 % BTS + 10 % SM), and P3 (85 % BTS + 15 % SM). The parameters observed were the colour, smell, consistency, pH, volume, motility, viability, and abnormalities of spermatozoa. The results showed that the characteristics were in the normal, creamy white semen colour, semen volume 3,6 ml, medium consistency, pH 6,3, typical SO bulls smell, sperm motility 85 %, mass activity +++, sperm concentration 1276 million/ml, live sperm 90,05 % and sperm abnormalities 6,15 % and quality there was no significant difference between the four treatments. The spermatozoa motility and viability in each treatment was able to which achieved on day 1. The characteristics of the spermatozoa of SO bulls were in the normal category. Meanwhile, BTS diluent with the addition of SKD was unable to maintain sperm quality.

EFFECT OF SWIM-UP AND GLASS WOOL TECHNIQUES, WITH ADDING ANTIOXIDANTS TO TRIS EXTENDER ON IMPROVING POST-CRYOPRESERVED SOME SEMEN ATTRIBUTES OF LOW SEMEN QUALITY FOR HOLSTEIN BULLS

This study was conducted to investigate the effect of swim-up and glass wool as sperm separation techniques with adding vitamin E and superoxide dismutase (SOD) to Tris extender on improving some post-cryopreserved semen quality for Holstein bulls. Low and good of semen were extended using Tris extender. Good semen quality (GSQ) was divided into 3 groups (L1; Tris extender, L2; 2 mM vitamin E, L3; 200 IU SOD) Low semen quality (LSQ) was divided into two main groups, and subdivided into 3 sub-groups (L4; Tris extender, L5; 2 mM vitamin E, L6; 200 IU SOD). In the second main group, swim-up and glass wool techniques were used with adding vitamin E and SOD and subdivided into 3 sub-groups with each technique, and referred to L7, L8 and L9 for swim-up technique and L10, L11 and L12 for glass wool technique. Improving sperms’ cell motility and live sperm and reducing total sperms’ abnormalities percentages of low semen quality were noticed using swim-up and glass wool techniques were used with adding vitamin E and SOD. In conclusion, glass wool filtration separates out dead, abnormal and immotile sperm cells, a good sperm harvested by this technique.

Honey Improves Sperm Parameters in High Cholesterol Diet-Fed Male Rabbits

Overconsumption of a high-energy diet has a negative impact on sperm motility, morphology, vitality and concentration. Hence, this study aims to investigate the effects of honey on sperm parameters of high cholesterol diet-fed rabbits and compare its effects with atorvastatin. Forty-eight male New Zealand white rabbits were assigned into 6 groups: Control (C): commercial pellet; CH: commercial pellet and 600 mg/kg/day Trihoney; HCD: 1% cholesterol diet; DH1: 1% cholesterol diet and 300 mg/kg/day Trihoney; DH2: 1% cholesterol diet and 600 mg/kg/day Trihoney; DAt: 1% cholesterol diet and 2 mg/kg/day atorvastatin. After 12 weeks, each rabbit was anaesthetized. Sperm were obtained from the cauda of the epididymis. Percentages of progressive (PM) and total motility (TM) were assessed using light microscope. Vitality and morphology were assessed using Eosin-Nigrosin stain. Sperm concentration was calculated using haemocytometer. Administration of 1% cholesterol diet reduced the percentages of PM, TM and normal sperm. Treatment with atorvastatin reduced the percentages of PM, TM, live and normal sperm. A marked reduction in sperm concentration was detected in the HCD and DAt groups. Trihoney groups expressed significantly higher percentages of PM, TM, normal sperm, live sperm and sperm concentration than the HCD and DAt groups. These results indicate that Trihoney has the potential to minimize the negative impacts of a high cholesterol diet on sperm parameters.

Flow-cytometric analysis of membrane integrity of stallion sperm in the face of agglutination: the “zombie sperm” dilemma

Purpose To define the effect of sperm agglutination, associated with incubation under capacitating conditions, on accuracy of membrane assessment via flow cytometry and to develop methods to mitigate that effect. Methods Sperm motility was measured by CASA. Sperm were stained with PI-PSA or a novel method, LD-PSA, using fixable live/dead stain and cell dissociation treatment, before flow-cytometric analysis. Using LD-PSA, acrosome reaction and plasma membrane status were determined in equine sperm treated with 10 μm A23187 for 10 min, followed by 0, 1, or 2 h incubation in capacitating conditions. Results Using PI-PSA, measured membrane integrity (MI; live sperm) was dramatically lower than was total motility (TMOT), indicating spurious results (“zombie sperm”). Sperm aggregates were largely of motile sperm. Loss of motility after A23187 treatment was associated with disaggregation and increased MI. On disaggregation using LD-PSA, MI rose, and MI then corresponded with TMOT. In equine sperm incubated after A23187 treatment, as the percentage of live acrosome-reacted sperm increased, TMOT decreased to near 0. Conclusion Flow cytometry assesses only individualized sperm; thus, agglutination of viable sperm alters recorded membrane integrity. As viable sperm become immotile, they individualize; therefore, factors that decrease motility, such as A23187, result in increased measured MI. Disaggregation before assessment allows more accurate determination of sperm membrane status; in this case we documented a mismatch between motility and live acrosome-reacted equine sperm that may relate to the poor repeatability of A23187 treatment for equine IVF. These findings are of profound value to future studies on sperm capacitation.

New Sperm Morphology Analysis in Equids: Trumorph® Vs Eosin-Nigrosin Stain

The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph® using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates from stallions and donkeys were used. After semen collection and dilution, an aliquot was placed on the slide and introduced in the Trumorph® device. Then observation was made with a 40x objective and negative phase-contrast microscope. Another aliquot was stained using eosin-nigrosine stain and viewed using 100× magnification. Well-formed sperm were observed, and different abnormalities were identified using Trumorph®. The use of eosin-nigrosin staining method and Trumorph® led to the same results and both techniques can be used for stallion and donkey sperm morphological analysis. However, considering the fact that Trumorph® uses living sperm helps prevent sperm cell alteration during sample preparation. Therefore, Trumorph® can be a good alternative to the conventional staining method, which provides a quick test on live sperm.

Effect of egg yolk-based extender and seminal plasma removal on sperm viability of cooled donkey semen

Developing effective cooled semen protocols is essential to increase pregnancy rates and reproductive efficiency in donkeys. This study aimed to evaluate the effect on sperm kinetic parameters and membrane integrity in cooled donkey semen diluted with defined milk proteins extender with 1% or 2% of egg yolk and the removal of seminal plasma. Twenty-four ejaculates from six jackasses were collected. Each ejaculate was divided into four aliquots that were diluted in extender with 1% (EY1) or 2% (EY2) egg yolk. One sample from each group was centrifuged, seminal plasma was removed (CEY1, CEY2 groups, respectively), and the samples were then refrigerated at 5 °C for 24 h. Fresh and cooled semen samples were assessed for sperm motility, morphology, and plasma membrane integrity. Total motility, progressive motility, sperm kinetic parameters, or live sperm cells were not statistically different when semen was cooled with an extender supplemented with 1% or 2% of egg yolk. Seminal plasma removal does not affect total motility or sperm kinetic parameters. However, progressive motility decreased (P<0.05) when semen was extended with 2% of egg yolk and seminal plasma was removed. Membrane integrity was affected (P<0.05) in centrifuged samples. In conclusion, the obtained results suggest that there is no difference in sperm kinetics and membrane integrity when 1% or 2% of egg yolk was added to the Equiplus(R) extender. Also, the removal of seminal plasma by centrifugation did not have any beneficial effect on cooled donkey semen. Further studies are needed to relate these results with in vivo fertility tests with cooled donkey semen.

Quality of deconserved bull sperm for the action of nanosuccinates Zn, Cu and Mn in the diluents

The purpose of this work was to compare effect of different doses of trace elements such as Cu2+, Zn2+ and Mn2+ that have been included as nano succinates into lactose-yolk-glycerol medium for cryopreservation of bull sperm and some physiological and biochemical sperm parameters assessment before and after cryopreservation. In this research each fresh ejaculate obtained from 4 bulls has been divided into parties consisting a control sample and its experimental counterparts. Control samples were diluted with industrial lactose-yolk-glycerin diluent only but their experimental counterparts were diluted and supplemented with nano acquacuccinates of Cu, Mn and Zn as solutions at concentration 2–5 g/l but different doses of 0.005, 0.01 and 0.05 mg/ml. When ejaculates were taken, the following physiological parametres of ejaculate quality were established: volume (ml), sperm concentration (billion/ml), live sperm count (%) and dynamic sperm count (CASA) and survival (h); content of total protein, respiratory activity of sperm, activity of enzyme markers of fertilizing ability — succinate dehydrogenase (SDH, units) and cytochrome oxidase (CHO, units) in diluted ejaculates with introduced minerals. After the ejaculates were diluted, semen was equilibrated for three hours at 4°C and frozen in a container (7 min over nitrogen vapor followed by immersion in liquid nitrogen). The semen was thawed in a water bath at 38°C for 20 seconds. The above physiological and biochemical parameters of the sperm of the bulls were redetermined immediately after thawing. Spermatozoa concentration in diluted bull sperm was 8.3% of the initial or ejaculate diluted 12-fold according to technological requirements (P<0.001). The number of live sperm decreased by 12.6% compared to fresh sperm (P<0.05), and the survival of sperm during incubation decreased by 6.8% for 7.4 hours. Total protein content in 100 ml of sperm decreased by 41.3% after dilution compared to fresh ejaculate (P<0.001). Respiratory activity decreased by 11.8% after the ejaculates was diluted. Succinate dehydrogenase activity decreased by 10.7% and cytochrome oxidase activity by 13.0%. In thawed bull sperm the respiratory sperm activity is higher in counterparts when 0.05 mg/l Zn2+, 0.05 mg/l Cu2+ and 0.05 mg/l Mn2+ are added to the medium. Enzyme activity at the same doses was higher. The highest activity among these groups of succinate dehydrogenase was at 0.05 mg/l Zn2+ (P<0.05) added to the cryopreservation medium, and the lowest at 0.01 mg/l Mn2+. Cytochrome oxidase activity was highest when 0.05 mg/l Cu2+ was added to the cryopreservation medium. The optimum concentrations of nanosuccinates that ensure the normalization of oxidation processes in the diluted bull sperm are: 0.05 mg/l Mn2+, 0.05 mg/l Cu2+ and 0.05 mg/l Zn2+. The higher concentration of metal nano succinates in the diluent inhibits the respiratory sperm activity and reduces the activity of succinate dehydrogenase and cytochrome oxidase. Similar effect has been estimated in dynamic performance of spermatozoa after thawing.