Clinical evaluation of chemo-sterilization through histomorphology and hormonal changes in bucks

The study was aimed to evaluate methods for nonsurgical castration of Black Bengal bucks by intra-testicular injection of calcium chloride, sodium chloride, citric acid solutions or sterile deionized water. Twelve healthy bucks were randomly allotted to groups A, B, C, and D, consisting of three bucks each. The local anesthetic, 2% lidocaine hydrochloride, was infused into the spermatic cord of each buck, followed by bilateral intra-testicular injections of 30% calcium chloride (CaCl2), 25% sodium chloride (NaCl), 50% citric acid (C6H8O7) solutions, and sterile deionized water dosed at 2 ml per testis in groups A, B, C and D respectively. To evaluate the efficacy of chemical agents on the inactivation of testes, clinical parameters, changes in scrotal circumference, testicular fine needle aspiration (TFNA), histopathology and serum concentration of testosterone and LH were monitored. A significant decrease in the scrotal circumference was observed between the intra-testicular injection and day 14 in all the bucks. Absence of spermatogenic cells and spermatozoa in the testicular biopsy was observed on day 14 post injection in the bucks, except for one in group C. Histopathology revealed massive destruction of seminiferous tubules and disorganization of the testicular parenchyma. Serum testosterone concentration declined significantly on day 14 compared with day 0. Consequently, the gradual elevation in serum LH concentration was significant. Thus, intra-testicular injections of CaCl2 and NaCl were more effective than C6H8O7 in inducing chemical-based nonsurgical castration

Monitoring Vaginal Electrical Impedance in Bangladeshi Water Buffaloes During Postpartum Period

The achievement of early cyclicity and pregnancy after calving are of vital importance for sustainable buffalo farms, for which proper detection of oestrus and correct time of AI are necessary. Vaginal electrical impedance (VEI) observation using heat detector was used to identify estrus in buffalo cows during post-partum period. In the first group, 12 buffalo cows with post-partum>300 days were synchronized with OVSYNCH protocol and VEI was recorded at different stages of synchronization protocol. VEI values decreased significantly (P < 0.01) during second administration of GnRH in both pregnant and non-pregnant buffalo cows and it was indicative for the presence of oestrus. Higher VEI values were obvserved at second administration of GnRH and AI in non-pregnant group than the pregnant group. In second group, 8 buffalo cows with a post-partum period 40 to 96 days were used to record VEI daily. Lower VEI values (260-350Ω)  were observed during oestrus and then started to increase at higher level after oestrus. The day difference between two successive lower/higher values of "VEI waves" ranged from 17-25 days, which might indicate the oestrous cycle in buffalo cows. Moreover, physical signs such as colour of vulva, mucus were less intensive in buffalo cows of second experiment. The study suggests that the measurement of vaginal impedance could serve as an indicator of the oestrus in buffalo cows and daily impedance measurements are necessary to confirm the stages of the estrous cycle

PROTEIN PROFILES AND BIOCHEMICAL CHARACTERISTICS OF SEMEN: INFLUENCE ON FROZEN-THAWED SPERMATOZOAL QUALITY IN RAMS (OVIES ARIES) AND ALPACAS (VICUGNA PACOS)

Seminal plasma (SP) is a complex biological fluid and is known to act as a nutritive-protective medium for spermatozoa suspended in it. Some of SP components are very important for sperm metabolism, as well as sperm function, survival and transport in the female reproductive tract. Remarkably little is known about biophysical and biochemical makeup of SP. Specially in camelids, the origin, composition and function of SP remain a mystery. Therefore, a series of experiments was conducted to study the biochemical composition of SP in ram and alpaca, and their possible relations with fresh semen quality and freezability. In the first experiment, as a basic study, we used two successive ejaculates collected from Sarda rams to observe the effect of ejaculate types (single and pool) on the freezability of spermatozoa. All samples cryopreserved using TRIS-citrate-egg yolk extender with 7% glycerol. We observed higher percentage of motile spermatozoa (P<0.05) with a highest motility score in post-thaw spermatozoa of pool of first and second ejaculates. In experiment two, ejaculates collected from Veneto endangered sheep breeds to study biochemical composition including Protein Profiles (PP) of SP in ejaculates and individual ram. Ejaculates were cryopreserved dividing into eight treatment groups using TRIS-based and skim milk-based extenders, and both fresh and post-thaw semen were evaluated for plasma membrane integrity with double fluorescence staining and plasma membrane architecture (phosphatidylserine translocation) with Annexin V assay along with routine semen parameters. Correlation between seminal parameters of fresh and cryopreserved semen with biochemical components of SP were statistically examined. Results of this experiment revealed ejaculate variation, more markedly, individual variation in SP composition and demonstrated that pooled (first and second) ejaculates could maintain sperm heterogeneity better when preserved with TRIS-based extender. Moreover, we observed negative but significant (P<0.01) correlation of total protein, Mg, P, and positive correlation (P<0.05) of protein bands of 75 kDa with apoptosis marker free viable sperm. In third experiment, caudal epididymal sperm collected from Veneto endangered sheep breed, were cryopreserved with TRIS- based and milk based extender to observe post-thaw sperm characteristics, and results demonstrated that post-thaw sperm quality of was inferior to that of ejaculated spermatozoa. In fourth experiment, we observed that diet could affect some SP components, especially, enzymes and energy profiles, and semen quality in terms of, semen volume, sperm concentration and sperm motility in alpaca. In experiment five, alpaca semen was cryopreserved with four types of extenders and both fresh and post-thaw semen were evaluated for plasma membrane integrity with double fluorescence staining and plasma membrane architecture (phosphatidylserine-PS translocation) with Annexin V. Results showed that TRIS based extender with 0.8% fructose could promote better post-thaw survivability of alpaca semen. we also demonstrated that there was correlation between biochemical composition of SP and both fresh and post-thaw semen quality. We found significant (P<0.01) correlation between semen viscosity, and percentage of apoptosis free sperm, Mg, P, total protein of SP. To our knowledge, this is the first report in the presence of apoptosis marker in alpaca viable sperm. However, our results confirm that biochemical composition and SP function varies with diet, ejaculate within male and between males and species. The composition of SP, therefore, is one factor that determines the suitability of individual male for semen cryopreservation and should also be taken into account in the preparation of medium during assisted reproductive techniques (ART) in order to preserve and possibly enhance post-thaw sperm fertilizing ability.Il plasma seminale è un fluido biologico complesso ed è conosciuto come una base nutritiva e protettiva degli spermatozoi. Alcuni componenti del plasma seminale sono di particolare importanza per il metabolismo spermatico, per la funzione, sopravvivenza e trasporto di queste cellule all'interno dell'apparato riproduttore femminile. Si conosce ancora poco come migliorare da un punto di vista biochimico e biofisico il plasma seminale. Nei camelidi, in particolare, l'origine non è conosciuta. Quindi, una serie di esperimenti sono stati condotti per studiare la composizione biochimica del plasma seminale negli arieti e negli alpaca e la relazione tra le caratteristiche biochimiche e la qualità e congelabilità spermatica. Nel primo esperimento, come studio di base, si sono usati due successivi eiaculati raccolti da arieti riproduttori di razza Sarda per osservare l'influenza del tipo di eiaculato sulla congelabilità degli spermatozoi. Si è osservato una più alta % di motilità spermatica nel post-scongelamento dei campioni raggruppati di primo e secondo eiaculato. Nel secondo esperimento, sono stati considerati eiaculati raccolti da arieti di razze venete a numerosità ridottissima per studiare il profilo biochimico, compreso quello proteico. Gli eiaculati sono stati suddivisi in 8 gruppi dividendo il campione in base al diverso trattamento di diluizione e di composizione del medium (TRIS o latte scremato). Sia i campioni freschi che quelli post-scongelamento sono stati valutati considerando l'integrità di membrana plasmatica utilizzando la tecnica in fluorescenza che considera la traslocazione della fosfotidilserina (Test dell'Annessina V) capace di rilevare il grado di apoptosi cellulare. I risultati di questo esperimento indicano una variabilità della composizione del plasma seminale, anche su base individuale, e che l'eterogeneità degli eiaculati (1° vs 2°) è meglio mantenuta quando si è usato il TRIS nel medium di diluizione. Inoltre , è stato osservato una correlazione negativa significativa tra proteine totali, Mg, P e spermatozoi con assenza di risposta al test di apoptosi. Un'altra interessante relazione positiva è tra la risposta negativa al test di apoptosi e proteine con peso molecolare di 75 kDa. Nel terzo esperimento, seme epididimale, raccolto da soggetti di razze veneto a numerosità ridotta, è stato trattato con TRIS o latte scremato per osservare le caratteristiche post-scongelamento degli spermatozoi. I risultati dimostrano come le qualità spermatiche sono inferiori rispetto agli spermatozoi di eiaculato. Nel quarto esperimento, si è osservato come la dieta può influenzare i componenti del plasma seminale ed in particolare il profilo energetico ela qualità seminale (volume, concentrazione, motilità) in alpaca. Nel quinto esperimento seme di alpaca è stato trattato con 4 differenti tipi di medium di diluizione ed il miglior risultato si è ottenuto utilizzando TRIS con lo 0,8% di fruttosio. Si sono messe in evidenza numerose interessanti correlazioni tra viscosità, % di cellule negative al test dell'apoptosi, Mg, P, proteine totali. In generale si può affermare che la composizione biochimica e la funzione del plasma seminale variano con la dieta e l'eiaculato anche per lo stesso soggetto e tra specie animali. La composizione è un fattore che determina l'utilizzo del singolo soggetto per la conservazione di materiale genetico è dovrebbe esser preso in considerazione per migliorare le tecniche di preparazione di medium di mantenimento o diluizione pre-congelamento per ottimizzare le tecniche di riproduzione assistita negli animali da reddito, per migliorare i programmi di conservazione di razze in pericolo e per migliorare la capacità fertilizzante post-scongelamento

Seminal plasma of ruminants: and essential attribute to spermatozoa

The biochemical composition of seminal plasma (SP) is very complex and variable among species. Advances in reproductive technologies reveal SP as a nutritive-protective medium for sperm cells suspended in it and some of SP components are very important for sperm metabolism, as well as, sperm function, survival and transport in the female reproductive tract. Biochemistry of SP is a relatively modern, but rapidly expanding field of research, particularly the biological significance of various bio-chemical constituents of SP. However, the identification of active factors in SP and mechanisms by which they act, are not sufficiently understood in different species. Especially in camelids, the origin, composition and function of the viscous component of SP remain a mystery and only some biochemical, morphological characteristic of SP have been described. This review focuses on our current understandings of physiological and biochemical properties of SP in ruminants and camelids

Seminal plasma: an essential attribute to spermatozoa

The biochemical composition of seminal plasma (SP) is very complex and variable among species. Advances in reproductive technologies reveal SP as a nutritive-protective medium for sperm cells suspended in it, and some SP components are very important for sperm metabolism, as well as sperm function, survival, and transport in the female reproductive tract. Biochemistry of SP is a relatively modern but rapidly expanding field of research, particularly concerning the biological significance of the various biochemical constituents of SP. However, identification of active actors in SP and mechanisms by which they act are not sufficiently understood in different species. Especially in camelids, the origin, composition, and function of the viscous component of SP remain a mystery, and only some biochemical and morphological character- istics of SP have been described. In this review, we focus on the current understanding of physiological and biochemical properties of SP in ruminants and camelids

Semen quality, total protein content and protein profiles of seminal plasma of alpaca (vicugna pacos) fed different diets

Advances in reproductive technologies reveal seminal plasma (SP) as a nutritive- protective medium for spermatozoa suspended in it. This complex physiological fluid contains various organic and inorganic components among which, a variety of proteins adsorb into plasma membrane during epididymal maturation and at ejaculation, affecting spermatozoal membrane stability and functions including fertilization. The potential influence of SP proteins on male reproduction has drawn attention because of their association with semen freezability and breeding scores in ruminants. Despite the relevance of SP proteins with fertility, studies on their precise natures and factors affecting their quantities and qualities are limited in alpacas. Therefore, this study was conducted to evaluate changes in total protein (TP) content and protein profiles (PP) of SP along with semen quality of alpacas maintained with different diets. Five alpaca males were used to collect semen with an artificial vagina. Semen was collected once in a week, over a period of 6 months from March to September, 2009. The study period had four sessions with four types of diets: I.hay; II.hay+ pasture grazing; III. Sheep concentrate (gross protein 16%, lipids 2.7%, fiber 10%, +pasture grazing; IV. horse concentrate (gross protein 12%, lipids 3%, fiber 11%,) + pasture grazing. After proper evaluation of semen quality, samples were centrifuged (3500 r.p.m., 40 min) to separate SP to perform biochemical study including gel electrophoresis. The SDS electrophoresis was performed using two concentrations of polyacrylamide (14% and 16%) in the gel of separation. The gels were colored with cromassie brilliant blue and the intensity was analyzed with Sante Dicom Viewer and Image J program. In this study there were great variations in TP and PP of SP along with semen quality of individual alpacas. TP (g/dl) were 4.5\ub14.04, 3.21\ub13.08, 3.06\ub12.93 and 6.38\ub12.83 in the sessions I, II, III and IV, respectively. Motility and concentration of spermatozoa were lower (18.5\ub18.8%, 10.44\ub123.35X106/ml respectively) during session IV. Assessment of the electrophoretic profiles of SP proteins showed a total of 7 bands having molecular weights from 200 to 14.00 kDa. Surprisingly, all bands were not present in all alpacas SP. Semen from males fed a high quality diet markedly differed and showed protein bands with molecular weights from 10 to 14 kDa and 31 to 45 kDa compared to semen of the other males. Moreover, there were significant changes in the percentage and quantity of similar protein profiles during the four sessions. Protein fractions of low molecular weights (14.40 to 31.00 kDa) were highest during session II and this fraction showed a positive correlation with sperm motility and concentration throughout the study. These results demonstrate that there are marked changes in semen quality, TP and PP of SP of alpaca depending on diets. In ruminants, SP proteins vary with individual animal, season of collection, temperature, nutrition and stress, and are originated from seminal vesicles, which are normally absent in camelides. The origin, composition and function of SP proteins remain a mystery in alpacas. For successful preservation of alpaca semen we should define good quality semen before processing by determining the biological and biochemical makeup of SP. This study could help us to define different protein profiles in alpaca SP and could lead us to find out functions and the specific sources of these proteins in future