39 Nanowater enhances cryoprotective effects of glycerol during ram semen freezing

2020 ◽  
Vol 32 (2) ◽  
pp. 145
Author(s):  
J. Szymanowicz ◽  
M. Murawski ◽  
T. Schwarz ◽  
P. Bartlewski

It was suggested that unique physicochemical properties of nanowater (NW, water declustered in the cold plasma generator), such as a low dielectric constant and high diffusivity, might ameliorate ram semen freezing by improving bioavailability of extender constituents. This study was undertaken to evaluate the effects of NW added to glycerol-containing semen extenders on the characteristics of frozen-thawed ram semen. A total of 18 ejaculates collected from 6 Olkuska breed rams during the breeding season were divided into six equal portions. The ejaculates were frozen in the modified fructose-skimmed milk-egg yolk Kareta extender containing 3 or 7% glycerol and diluted in deionized water (control extenders; Aqua Purificata, Prolab; C3% and C7%, respectively) or NW (declustered for 15min (NW15’) or 30min (NW30’); Nanotechnology Systems) to a final concentration of 800×106 spermatozoa/mL. They were placed in 0.25-mL plastic straws and frozen in liquid nitrogen. The two declusterization times were chosen on the basis of previous laboratory tests and fertility trials yielding the best results in terms of semen quality post-thawing and pregnancy rates after AI. All semen samples were evaluated for sperm concentration, progressive motility (Sperm Class Analyzer), and morphological defect rates (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of alanine transferase, alkaline phosphatase, and aspartate aminotransferase were measured, along with the proportions of apoptotic, necrotic, and live spermatozoa determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson) at 0 and 1h post-thawing. Data were analysed by one-way analysis of variance and Holm-Sidak method using SigmaPlot statistical software (Systat Software Inc.). The proportion of spermatozoa with mid-piece defects was significantly (P<0.05) reduced in NW15’-3% compared with C3% (1.3±0.3% vs. 4.0±06%; mean±s.e.m.). The proportion of necrotic spermatozoa 1h after thawing was greater (P<0.05) in C7% compared with NW30’-7% (20.7±0.4% vs. 17.6±1.0%), whereas the proportions of live cells detected immediately (0h) and 1h after thawing were greater (P<0.05) in NW30’-7% than in C30’ (0 h: 54.0±0.6% vs. 50.4±1.2%; 1 h: 54.4±1.2% vs. 59.2±0.4%, respectively). The mean survival time of spermatozoa was greater (P<0.05) in extenders dissolved in NW30’compared with their respective controls (NW30’-3% vs. C3%: 215±4min vs. 189±6 min; and NW30’-7% vs. C7%: 242±8min vs. 217±4min, respectively). Alkaline phosphatase concentrations in extenders prepared with NW30’ were lower (P<0.05) compared with the control groups (NW30’-3% vs. C3%: 3105±231IU vs. 4490±458 IU; and NW30’-7% vs. C7%: 2516±128IU vs. 3956±263IU, respectively). Our results indicate that NW significantly improves the effects of glycerol on ram sperm viability post-thawing, with the reduction in sperm necrosis/overall enhancement of sperm survivability being greater with 7% compared with 3% of glycerol in semen extender.

2020 ◽  
Vol 32 (2) ◽  
pp. 138
Author(s):  
M. Murawski ◽  
J. Szymanowicz ◽  
T. Schwarz ◽  
P. Bartlewski

This study examined the effect of genotype and extender diluent on the characteristics of frozen-thawed ram semen. Twenty ejaculates collected from 10 rams aged 4-12 years (2 Polish Lowland (PON), 4 synthetic line BCP (Berrichon du Cher×Charolais×PON/Polish Merino), and 4 SCP (Suffolk×Charolais×PON/Polish Merino) in mid-breeding season were divided into six equal portions each and frozen in semen extender prepared by mixing commercial Triladyl extender (MiniTub GmbH) with deionized water (Aqua Purificata, Prolab) or nanowater (NW; deionized water declusterized in a cold plasma generator for 15, 30, 45, 60, or 90 minutes (15’, 30’, 45’, 60’, or 90’, respectively); Nanotechnology Systems) and Gallus domesticus egg yolk (1:3:1 vol/vol/vol). All semen samples were evaluated for progressive motility (Sperm Class Analyzer) and sperm morphology (Nikon Eclipse 80i microscope, Nikon Corp.). In addition, the ex situ survival time at 37°C and extender content of ALT (alanine transferase), ALP (alkaline phosphatase), and aspartate aminotransferase were measured, and the numbers of apoptotic, necrotic, and live spermatozoa were determined post-thawing with flow cytometry (BD Accuri™ C6 Plus, Becton Dickinson). Data were analysed by two-way analysis of variance and least significant difference test (SigmaPlot, Systat Software Inc.). The PON exceeded (P<0.05) SCP rams in the occurrence of mid-piece and double tail defects using NW30’ and NW45’ extenders. The proportion of spermatozoa with proximal droplets was greater (P<0.05) in PON than in SCP or BCP rams with NW15’ and NW30’ extenders, respectively, but the percentage of sperm with detached heads was greater (P<0.05) in BCP and SCP compared with PON using NW15’ extender. Overall, spermatozoa in PON rams’ semen had survival times greater by 25-50min (P<0.05) compared with those for semen from BCP and SCP synthetic lines. The ALT levels were significantly elevated in SCP compared with PON and BCP rams only in NW90’ extender. The numbers of necrotic spermatozoa were greater (P<0.05) in SCP and BCP compared with PON rams, and the numbers of apoptotic spermatozoa were greater (P<0.05) in BCP than in SCP rams with NW45’, NW60’, and NW90’ extenders. Live spermatozoa were more prevalent (P<0.05) in PON than in BCP rams in NW45’ extender post-thawing. To summarise, semen from PON rams had higher survival times and fewer spermatozoa with detached heads but more mid-piece defects and sperm with double tails post-thawing compared with BCP and SCP breeds. The number of necrotic cells was greater in semen from BCP and SCP rams compared with PON. It can be concluded that breed-related differences in post-thaw sperm morphology were apparent in extenders containing NW15-60’, whereas the variations in cell necrosis/apoptosis were primarily seen with the use of NW45-90’ extenders.


2013 ◽  
Vol 25 (4) ◽  
pp. 683 ◽  
Author(s):  
N. S. Juyena ◽  
J. Vencato ◽  
G. Pasini ◽  
I. Vazzana ◽  
C. Stelletta

The aim of the present study was to evaluate the biochemical composition of seminal plasma, along with semen quality, of alpacas maintained on different diets (hay; hay + pasture grazing; pasture grazing + sheep concentrate; pasture grazing + horse concentrate; Periods 1–4, respectively). Alpacas (n = 5) were fed the four different diets for a period of 6 weeks each. During the period of feeding of each diet, semen was collected using an artificial vagina to determine its volume, viscosity, sperm concentration and sperm motility. Moreover, testicular volume and body condition score were evaluated. Seminal plasma was analysed biochemically to measure total protein, triglyceride, cholesterol, γ-glutamyl transferase, alanine aminotransferase (ALT) and alkaline phosphatase levels. Protein profiles were investigated using one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. There was high variability in semen parameters between different males maintained on the same diet. Semen volume increased significantly (P < 0.05) when alpacas were fed diets containing commercial sheep and horse concentrates. In contrast, sperm concentration and motility decreased significantly (P < 0.05) from Period 1 to Period 4. Dietary changes had no effect on viscosity. Significant reductions were seen in triglyceride and cholesterol content, as well as γ-glutamyl transferase, ALT and alkaline phosphatase concentrations, from Period 1 to Period 4. Regardless of experimental period, a wide variation was seen in seminal plasma enzyme concentrations between alpacas, whereas diet had no effect on glucose and total protein concentrations in the seminal plasma. Eight protein bands, with molecular weights ranging from 200 to 14 kDa, were considered in electrophoresis gel after image analysis. Proteins fractions of the 14-kDa (total protein express in md dL–1 with a molecular weight of 14-kDa, TP8) and 21-kDa (total protein express in md dL–1 with a molecular weight of 21-kDa, TP7) bands were not present in all samples of alpaca seminal plasma. There were no significant changes in the concentration of any protein fractions during the four periods. Moreover, the protein fraction of the 60-kDa (total protein express in md dL–1 with a molecular weight of 60-kDa, TP3) band was the most prevalent in all periods. These results demonstrate that there are marked changes in semen quality, as well as some parameters related to the composition of alpaca seminal plasma, that are dependent on diet, which may indicate the need for specific diet formulation to improve reproductive performance. We hypothesise that, in alpacas, the mechanisms underlying the changes in some reproductive traits in response to feeding regimens could be related to changes in the endocrine–gonadal system.


1970 ◽  
Vol 7 (2) ◽  
pp. 305-308 ◽  
Author(s):  
S Pervage ◽  
MR Hassan ◽  
M Ershaduzzaman ◽  
MAMY Khandoker

An experiment was undertaken to determine the conception rate of native sheep by using Artificial Insemination with liquid ram semen. The semen was collected from ram using artificial vagina and the was stored in a refrigerator (4°C) for three days. The volume of semen was extended with egg yolk citrate diluter. A total of 63 ewes were inseminated with stored liquid semen collected from 15 rams by AV method. The total number of spermatozoa, live-dead, normal-abnormal, sperm motility and the pH was observed regularly. The average semen volume per ejaculate was 0.76-1.00ml and the sperm concentration was 2.37x109 - 4.30x109 per ejaculate. The number of normal spermatozoa and the pH was almost similar irrespective of days of storage. Number of live spermatozoa and the sperm motility were reduced with the increasing age of semen. The average conception rate (%) was obtained as 63.61, 61.90, 52.38 and 47.61 with sperm in zero, 1st, 2nd and 3rd day storage respectively. Keywords: Preservation; Liquid semen; Quality; Artificial insemination; Sheep DOI: 10.3329/jbau.v7i2.4739 J. Bangladesh Agril. Univ. 7(2): 305-308, 2009


2007 ◽  
Vol 19 (1) ◽  
pp. 128
Author(s):  
C. Tamargo ◽  
C. Díez ◽  
J. De La Fuente ◽  
M. Carbajo ◽  
J. M. Benito ◽  
...  

The need to conserve farm animal biodiversity is accepted by many countries through the ratification of the convention of biological diversity, and sperm quality is known to be an important criterion in the evaluation of breeding soundness. The aim of this work was to characterize the semen of a local breed of ponies 'Asturcon' (maintained free over the mountains all year around) before its incorporation into a germplasm bank. Semen was obtained from six stallions (6–17 years of age) using an artificial vagina, 3 days/week, during 12 weeks. Immediately after collection, gel-free semen was evaluated for volume, sperm concentration, and motility. Semen motility was again evaluated after equilibration/refrigeration. For evaluation of individual (IM) and progressive motility (PM) rates, semen was diluted (20 � 106 spermatozoa/mL) and analyzed with a CASA System (SCA; Microptic S.L., Barcelona, Spain). Five fields per sample were evaluated (minimum 500 spermatozoa/sample) under a phase contrast microscope (100�). Semen samples were subjected to a hypo-osmotic swelling test (HOS) test to detect the presence of swollen tails in a 100 mM citrate–fructose solution. Percentages of altered acrosomes and morphological abnormalities were determined by counting 100 spermatozoa (1000�). Then, semen was diluted and centrifuged for 10 min at 600g. After the supernatant was discarded, the pellet was re-suspended in freezing medium (skim milk extender containing 2% egg yolk and 2.5% glycerol) to a final concentration of 100 � 106 spermatozoa/mL, and equilibrated/cooled (60 min) to 4�C. Statistical analysis was carried out by means of the GLM and CORR procedures and Duncan test for means (SAS Institute, Inc., Cary, NC, USA). A significant effect between males (P &lt; 0.05) on semen quality, such as volume of the ejaculate, sperm concentration, and morphological abnormalities, were detected among stallions. On the other hand, positive and significant correlations were found between the sperm motility immediately after collection and after equilbration/refrigeration (r = 0.73; P &lt; 0.05); moreover, sperm motilities (both fresh and refrigerated) correlated with the results of the HOS test (r = 0.56; P &lt; 0.001, and r = 0.27, P &lt; 0.05, respectively). These preliminary results confirm that the sperm of the Asturcon ponies breed can be collected and will survive the equilibration/refrigeration procedures. Conservation and development of local breeds is important because they represent a unique source of genes for improving health and performance traits of industrial breeds. However, complementary studies on the ability of the stallion sperm to survive freezing/thawing procedures in rates higher than 30% are needed to ensure that genetic banks are correctly created. This work was performed in collaboration with ACPRA and Dep�sito de Sementales de Santander (Spain), and supported by RZ2004-00031-C02-01.


2021 ◽  
Vol 44 (3) ◽  
pp. 84-88
Author(s):  
K. U. Anoh ◽  
P. P. Barje ◽  
G. I. Iyeghe-Erakpotobor ◽  
G. N. Akpa

The objective of the study was to evaluate the effect of baobab fruit pulp meal on semen characteristics and morphology of rabbit bucks during hot season in Nigeria. A total of 25 bucks of 10-12 months old were used. There were five (5) rabbits per group grouped as follows; T1 (control) and T2 -T5 (Diets with graded levels of baobab fruit pulp meal BFPM at 2.5, 3.5, 4.5 and 5.5% inclusion levels). The experiment lasted for nine weeks. All managerial practices were duly observed. Semen was collected with the help of artificial vagina, and the semen samples were evaluated for ejaculate volume (ml), semen pH, semen colour, sperm motility (%) and sperm concentration (x 106 /ml). Sperm morphological parameters evaluated were live cells, dead cells, normal sperm, free tail and coil tail. It was observed from this study that 4.5% inclusion of baobab fruit pulp meal significantly (P<0.05) influenced semen volume, colour, motility, pH and concentration compared to 5.5% inclusion level and was similar to 2.5% and 3.5% inclusion levels, respectively. The treatment with 4.5% BFPM also significantly (P<0.05) improved the number of live cells (74.38%), reduced dead cells (25.62%) and increased number of normal cells (77.71%) compared to 65.67, 34.33, and 60.67% recorded by the control for live cells, dead cells and normal cells, respectively. This study revealed that 4.5% inclusion of BFPM improved semen quality during hot season.


2019 ◽  
pp. 141-145 ◽  
Author(s):  
R. Saha ◽  
S. Roychoudhury ◽  
K. Kar ◽  
A.C. Varghese ◽  
P. Nandi ◽  
...  

This study aimed at investigating the protective role of CoQ10 against cadmium (Cd)-induced reproductive toxicity in male rats. Adult male Wistar rats were exposed to an acute dose of Cd (25 mg/kg bwt; Cd group), Cd+CoQ10 (25 mg/kg bwt Cd+10 mg CoQ10; Cd-Q10 group) and distilled water (control) in vivo for 15 consecutive days and semen quality was assessed. A significant reduction was noted in sperm concentration, progressive motility, morphology and DNA integrity in both Cd- and Cd-Q10 groups in comparison to control indicating Cd-induced testicular lipid per oxidation (LPO) and decline in indigenous antioxidant defense system as measured by total antioxidant capacity (TAC) (p<0.05). However, simultaneous co-administration of CoQ10 along with Cd (Cd-Q10 group) was able to improve sperm concentration, motility, progressive motility, morphology, DNA integrity, and testicular TAC as well as lower LPO compared to Cd group (p<0.05). Results indicate that used dose of CoQ10 is capable of moderately ameliorating reproductive toxicity of Cd by improving semen quality and reducing testicular oxidative stress.


2019 ◽  
Vol 6 (1) ◽  
pp. 78
Author(s):  
Muhammad Ade Salim ◽  
Muhammad Nur Ihsan ◽  
Nur Isnaini ◽  
Trinil Susilawati

ABSTRAKAir kelapa muda varietas viridisdapat dijadikan pengencer aletrnatif semen cair bagi program IB di daerah minim sarana semen beku. Tujuan penelitian ini untuk menguji pengaruh penggunaan air kelapa muda viridissebagai bahan pengencer terhadap kualitas semen cair kambing Boer setelah didinginkan. Dilaksanakanselama 3 bulan di Laboratorium Fakultas Peternakan UBUnit SumberSekar,Malang. Metodenya yaitu eksperimen. Semen dari  3 pejantan Boer umur 3-5 tahun, dikoleksi seminggu sekali dengan VB. Air kelapa mudaviridis umur 5-7 bulan serta tris aminomethane sebagai kontrol. Didesain menggunakan Rancangan Acak Kelompok (RAK) dengan 2 perlakuan yaitu P0 (tris aminomethane + 10% KT) dan  P1 (air kelapa muda viridis + 10% KT) masing-masing diulang 10 kali. Data dianalisis dengan analisis Ragam (Anova) dengan software Genstat 18. Variabelnya yaitu motilitas individu, viabilitas dan abnormalitas. Hasil penelitian yaitu motilitas individu pada P1bertahan sampai 4 hari (40,5± 24,3%), viabilitas terbaik sampai hari ke-5 (42±24,6%), abnormalitas terendah di hari ke-7(1,31± 0,6). Kesimpulannya, Pengencer air kelapa muda viridis dapat mempertahankan kualitas semen cair kambing Boer selama 4 hari untuk motilitas dan 5 hari untuk viabilitas.Kata Kunci:pengencer, air kelapa, varietas viridisABSTRACTYoung viridis coconut water could be used as an alternative to liquid semen diluent for artificial insemination program in the area with limited facility for frozen semen production. This study evaluated the use of young coconut water as a diluent on liquid semen quality of Boer goat after cold storage. This study was carried out for 3 months at Sumber Sekar Laboratory, Faculty of Animal Husbandry, University of Brawijaya, Malang. The semen was collected from 3 Boer bucks aged at 3 to 5 years old. The semen collection was done once a week with the aid of artificial vagina. The diluents used were young Viridis coconut (5 to 7 months old) and tris aminomethane. The method used was an experiment in a randomized block design with 2 treatments and 10 replicates. The treatments used were T0: tris aminomethane + 10% egg yolk (control) and T1:  young Viridis coconut water + 10% egg yolk. Data were analyzed by analysis of variance using Genstat 18 software. The variables measured were sperm individual motility, viability, and abnormality. The results showed that the sperm individual motility in T1 survived up to 4 days (40.5± 24.3%), the best viability at 5 days (42.0±24.6%),  while the lowest abnormality at 7 days (1.31±0.6). It could be concluded that: 1. Tris aminomethane diluent has higher quality with the storage length up to 9 days, 2. Young Viridis coconut water diluent could preserve liquid semen quality of Boer goat up to 4 days for sperm motility and 5 days for sperm viability.Keywords: diluents, coconut water, viridis variety


Author(s):  
Amina Kumarovna Karamuldaeva ◽  
Andrey Mikhailovich Tikhomirov

The article studies the possibility to use glycerol as cryoprotectant, instead of dimethylsulfoxide for cryopreservation of sperm of inconnu ( Stenodus leucichthys Gueldenstaedtii, 1772). Investigations were carried out from 2015 to 2016 in the laboratory of the Southern Scientific Center, Russian Academy of Sciences, on the basis of the Astrakhan State Technical University. The material collected on the Alexander sturgeon hatcheries (the Astrakhan region) in the spawning period. Native sperm of 6 male inconnu species was used as a control means. The semen quality was determined in terms of moving activity (life time) of sperm after its activation by water. As the cryoprotectant there were used: base solution - 80%, sucrose - 1.71 g/l, mannite - 0.98 g/l, yolk - 10%, dimethylsulfoxide - 10% and base solution - 87%, sucrose - 1.71% g/l, mannite - 0.98 g/l, yolk - 10%, glycerol - 3 variants: 3; 5 and 10%. In order to provide the most complete penetration of cryoprotectants into the cells there were used electrostimulation of cell membranes. Equilibration time was 5 and 15 minutes. Thawing semen was performed in a water bath at a temperature of 38-40°C. For removing protectors from cells there was chosen a saline solution (0.7% NaCl) as isotonic solution. In tests using dimethylsulfoxide life activity of sex cells was 2 times lower than in tests with glycerol: 78 and 186.2 s at the end of equilibration and 52.3 and 128.9 s after thawing. Sperm showed maximum activity under 5% glycerol concentration during equilibration - 15 min. Concentration of 3% was insufficient, concentration of 10% was excessive, as it suppressed activity of sperm. Egg yolk which coagulated together with glycerol, making difficulty for observing, had to be excluded from the composition of cryoprotectant.


Author(s):  
Thea Emily Benson ◽  
Anne Gaml-Sørensen ◽  
Andreas Ernst ◽  
Nis Brix ◽  
Karin Sørig Hougaard ◽  
...  

Bisphenol A (BPA) is considered an endocrine disruptor and has been associated with deleterious effects on spermatogenesis and male fertility. Bisphenol F (BPF) and S (BPS) are structurally similar to BPA, but knowledge of their effects on male fertility remains limited. In this cross–sectional study, we investigated the associations between exposure to BPA, BPF, and BPS and semen quality in 556 men 18–20 years of age from the Fetal Programming of Semen Quality (FEPOS) cohort. A urine sample was collected from each participant for determination of BPA, BPF, and BPS concentrations while a semen sample was collected to determine ejaculate volume, sperm concentration, total sperm count, sperm motility, and sperm morphology. Associations between urinary bisphenol levels (continuous and quartile–divided) and semen characteristics were estimated using a negative binomial regression model adjusting for urine creatinine concentration, alcohol intake, smoking status, body mass index (BMI), fever, sexual abstinence time, maternal pre–pregnancy BMI, and first trimester smoking, and highest parental education during first trimester. We found no associations between urinary bisphenol of semen quality in a sample of young men from the general Danish population.


1999 ◽  
Vol 17 (1) ◽  
pp. 173-173 ◽  
Author(s):  
Peter Meidahl Petersen ◽  
Aleksander Giwercman ◽  
Steen W. Hansen ◽  
Jørgen G. Berthelsen ◽  
Gedske Daugaard ◽  
...  

PURPOSE: To elucidate the biologic association between germ cell neoplasia and testicular dysfunction, through investigation of Leydig cell function and semen quality in men with carcinoma-in-situ (CIS) of the testis. PATIENTS AND METHODS: We examined two groups of men, unilaterally orchidectomized for testicular cancer. Biopsy of the contralateral testis had showed CIS in a group of 24 patients and no evidence of CIS in the other group of 30 patients. Semen quality and serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were compared in these two groups of men after orchidectomy but before further treatment for testicular cancer. RESULTS: Significantly higher LH levels (median, 8.1 IU/L v 4.8 IU/L; P < .001) and generally lower testosterone levels (median, 12.5 nmol/L v 15.5 nmol/L; P = .13) were found in the CIS group. The proportion of patients with Leydig cell dysfunction was higher in the group of patients with CIS (11 of 24) than in the group of patients without (two of 30) (P = .01). Sperm concentration and total sperm count were significantly lower (P < .001) in patients with CIS (median, 0.03 × 106/mL and 0.10 × 106, respectively) than in patients without (median, 9.1 × 106/mL and 32 × 106, respectively), whereas the levels of FSH were significantly higher (P < .001) in the former group of men (median, 19.6 IU/L v 9.0 IU/L). CONCLUSION: Not only spermatogenesis but also Leydig cell function is impaired in testes with CIS. This impairment could be due to common factors in the pathogenesis of germ cell neoplasm and testicular dysfunction. Alternatively, CIS cells may have a negative impact on Leydig cell function.


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