Effect of Extenders Containing Glycerol and Egg Yolk on Motility and Viability of Chilled Ram Semen collected during Non-Breeding Season (original) (raw)
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This study aimed to evaluate the effect of alternative components of egg yolk in Tris extender and semen packaging methods during cryopreservation on sperm characteristics and antioxidant system in seminal plasma of frozen-thawed semen of Finnish Landrace rams. Semen was collected from 5 sexually matured Finnish rams (50-70 kg LBW and 2-4 years old) by artificial vagina once weekly for 7 weeks. Only semen with mass motility of ≥70% was pooled and diluted with Tris-citric extender containing 15% egg yolk (Tris-EY) or 1% soybean lecithin (Tris-SBL) or 2 mM butylated hydroxytoluene (Tris-BHT). Semen was extended at a rate of 1:5 (semen/extender) with three extender types. After semen extension, semen was placed for cooling in the refrigerator (5 o C) for 4 hours as equilibration period and packaged in 0.25 ml French straws or 0.25 ml pellets in liquid nitrogen. Semen was thawed at 37°C for 30 s. Semen was evaluated after dilution, equilibration and thawing, for progressive motility, livability and abnormality of spermatozoa and curled tail spermatozoa responded to a solution of osmolarity of 75 mOsm for 30 min. The concentration of total antioxidants (TAC), malondialdehyde (MDA) and lactic dehydrogenase (LDH) activity in post-thawed seminal plasma were determined. Results showed that sperm characteristics, including percentages of progressive motility, livability, abnormality and curled tail in post-diluted, post-equilibrated or post-thawed semen were not affected significantly by the type of extender. Progressive motility and curled tail percentages in post-thawed semen were higher (P<0.001; P<0.05) in straws than in pellets. Livability and abnormality percentages were insignificantly better at straws than in pellets. The recovery rate of motility and livability was higher at straws than in pellets. All sperm characteristics indicated insignificant effect of interaction between type of extender and semen packaging method. TAC was higher (P<0.05), while MDA concentration was lower (P<0.05) in Tris-SBL and Tris-BHT than in Tris-EY. The activity of LDH was insignificantly the highest in Tris-BHT than in other extenders. The TAC, MDA concentration and LDH activity in post-thawed semen were not affected significantly by semen packaging method. Effects of interaction between type of extender and semen packaging method on each of TAC, MDA concentration and LDH activity were not significant. The current study concluded the successful usage of Tris-SBL or Tris-BHT in comparing with the possible disadvantages of using egg yolk in Tris-based extender of ram semen.
Effect of different extenders and storage periods on motility and fertility of ram sperm
Macedonian Veterinary Review, 2015
The aim of this study was to test the effect of extenders containing different sugar in their composition on ram sperm motility and pregnancy rate of ewe’s following artificial insemination. Semen were collected from ten North-east Bulgarian fine-fleece breed and tested for quality. Semen was diluted with different extenders, with di- and trisaccharides. A series of experiments were repeated in triplicate. Total motility was determined by using Sperm Analysis (SCA, Microptic, Spain). A total of 200 North-east Bulgarian fine-fleece breed mature ewes were used for cervical insemination with a sperm dose at the concentration of 100 × 106spermatozoa. Pregnancies were diagnosed 60 days after AI by - a real-time ultrasonic scan device (Alloka SSD 500). In conclusion, our experiments demonstrated that higher sperm motility after storage at 4°C for 24 hours and 48 hours has a ram spermatozoa diluted with extender 1, with combination of disaccharides (sucrose and lactose) and trisaccharides ...
Reproduction in Domestic Animals, 2011
This study aimed at comparing in vitro, ultra-heat-treated (UHT) skim milk and INRA-96 Ò -based extenders supplemented or not with 5% egg yolk and ⁄ or 2% glycerol on sperm quality parameters along 72 h of preservation at 5°C, using a factorial design. Semen from six healthy mature Merino rams was pooled and extended in each medium using a split sample procedure (six replicates) and chilled. Subjective motility (SM) (%), membrane integrity (MI) (%) and uncapacitated spermatozoa (US) (·10 6 spermatozoa ⁄ AI dose) were used to assess the semen quality at 0, 12, 24, 48 and 72 h of preservation. UHT-based extenders yielded better (p < 0.05) SM and MI than INRA-96 Ò -based extenders (59.7% vs 57.9%; 60.2% vs 55.8%, respectively) but similar numbers of US (64.2% vs 62.3 · 10 6 sperm ⁄ AI dose, respectively) along the preservation time. Egg yolk-glycerol or just egg yolk as additives improved (p < 0.05) the results compared with the base extenders without additives or just with glycerol. The sperm parameters assessed decline slowly from 0 to 48 h, with a sharp decline (p < 0.05) at 72 h of preservation. In conclusion, UHT and INRA-96 Ò were similar as base extenders, and the addition of 5% egg yolk plus 2% glycerol or just 5% egg yolk improved the quality of ram semen preserved at 5°C, at least for 48 h. The combination of egg yolk-glycerol might provide extra protection in case of fluctuation of temperatures below 5°C, commonly seen under field conditions.
Impact of Conventional and Non-Conventional Extenders on Rams Semen Quality During Storage at 5ºC
The aim of the present study was to define the effect of different extender components (as conventional and non-conventional extenders) on rams' semen quality during storage at 5ºC for up to 6 days. Fertility rates of the extended ram spermatozoa stored at 5ºC for up one day were also studied. Four Rahmani rams were used to collect pooled ejaculates by artificial vagina. Semen was collected, evaluated and extended with five extenders included conventional and non-conventional extender ingredients. The conventional extenders were sodium citrate (E1), Tris (E2) and skimmed milk powder (E3)-based glucose-egg yolk. While, non-conventional extenders were salt of sodium chloride 0.9% (E4) and saline solution 0.9% intravenous infusion (E5) based glucose-egg yolk. The final extension rate was 1semen : 6 extender. The extended semen with different extenders media (E1, E2, E3, E4 and E5) were cooled to 5ºC and stored at this temperature for 6 days. After storage time (0, 1, 2, 3, 4, 5 and 6 days), the percentage of sperm motility (SPM), recovery sperm motility (RSM) and positive osmotic resistant (POR) were recorded. Also, fertility rate was carried out with liquid semen stored at 5ºC for up to one day using the best conventional and non-conventional extenders. Thus, twenty-two ewes were used to investigate fertility rate. Ewes were divided into two groups (11/group), the1 st and 2 nd groups were artificially inseminated with conventional (E2) or non-conventional (E5) extenders which obtained better semen quality than other extenders, respectively. The results indicated that, the cooling extended ram semen with each of E1, E2 or E5 extenders was significantly (P<0.05) higher the percentage of SPM, RSM and POR than those extended with E3 and E4 extenders during storage at 5ºC for up to 6 days. However, the cooling ram semen with E2 and E5 showed non-significantly parameters during cooled at 5ºC till 6 days. The statistical analysis of the data revealed a significant positive correlation between extender types and SPM, RSM and POR of ram spermatozoa during storage at 5ºC until 6 days. Surprisingly, fertility rate of ewes artificially inseminated with ram semen preserved at 5ºC for one day of storage performed to assess higher conception rate (84.62%) with E5 (as non-conventional extender) than conception rate (78.57%) with E2 (as conventional extender). Moreover, the superior litter size recorded 1.00 with ram semen extended with E5 compared to 0.86 with E2 extenders. Also, sex ratio of born lambs displayed female lambs 66.67 and 33.33% of male lambs with semen extended with E2 extender. Ram semen extended with E5 achieved female lambs 69.23% and male lambs 30.77%. It is therefore concluded that saline solution (0.9%) intravenous infusion (E5) based glucose-egg yolk intended to keep goodness fertility rate and litter size after AI of cooled semen at 5ºC.
Effect of different extenders for preservation of ram semen at 4°C
Indian Journal of Animal Research, 2016
The aim of this study was to investigate the effects of different extenders viz. Tris citric acid fructose egg yolk (TCFEY), Tris citric acid glucose egg yolk (TCGEY), Egg yolk citrate fructose (EYCF) and Egg yolk citrate glucose (EYCG) on the quality of ram spermatozoa during preservation at 4°C. Semen samples showing more than 3+ mass motility and 70% progressive motility were pooled and subsequently divided into four aliquots. Each aliquot was extended separately in four different extenders viz. TCFEY, TCGEY, EYCF and EYCG and stored at 4°C up to 72h. The quality of spermatozoa on the basis of percentage of sperm motility, live sperm, morphological abnormalities, intact acrosome and hypoosmotic swelling test (HOST) reacted spermatozoa was evaluated immediately after extension in particular extenders (0 h), 24 h, 48 h and 72 h after preservation at 4°C. The percent sperm motility was significantly…
Theriogenology, 2003
Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell 1 ; IMV, L'Aigle, France) free from additives of animal origin, containing two different ®nal glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 8C or at 15 8C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA)), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no signi®cant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell 1 (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 8C, although the results were not always statistically signi®cant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5±10%) concentrations of egg yolk and the addition of glycerol at 5 8C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell 1 containing 6.4% Theriogenology 59 (2003) 1241±1255
Turkish Journal of Veterinary & Animal Sciences, 2012
Th is study was conducted to evaluate the eff ects on sperm motility and abnormality in ram semen extended with skimmed milk (M), sodium citrate (SC), Tris (T), and Bioxcell® (B) aft er storage in liquid form at 4 °C and in frozen form. Ejaculates were collected from 3 Akkaraman rams by artifi cial vagina twice a week during the non-breeding season. Aft er pooling, each pooled ejaculate was split into 4 equal aliquots and diluted with skimmed milk (M), sodium citrate (SC), Tris (T), and Bioxcell® (B) extenders. Sperm motility was signifi cantly higher (P < 0.05) in M compared with B, SC, and T on the fi rst and second days (48 h) of storage. With regard to the percentage of total abnormal spermatozoa, the results in M were diff erent from (P < 0.05) from those of SC and T on the fi rst day (24 h) of storage but no diff erent from B. Diff erences among extenders were found to be signifi cant post-thawing for spermatozoa motility and the percentage of total abnormal spermatozoa. Th e total abnormality of semen diluted with M was signifi cantly lower than that observed in the other extenders. Consequently, it was found that skimmed milk was better than the other extenders in terms of the sperm parameters evaluated during liquid storage and post-thaw.
Effect of different extenders and storage temperatures on sperm viability of liquid ram semen
Theriogenology, 2002
Semen was collected with an arti®cial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 8C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a¯uorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac 1 and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were in¯uenced by storage time and extender, while sperm motility was the only evaluated parameter that was in¯uenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 8C seemed to in¯uence sperm viability parameters less than storage at 20 8C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate-and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 8C. Whether the differences found between the extenders will be re¯ected in the Theriogenology 57 (2002) 823±836 : S 0 0 9 3 -6 9 1 X ( 0 1 ) 0 0 6 8 3 -5 fertility results after AI is yet unknown and needs to be further studied. #
ADDITIVES USED IN EXTENDERS TO IMPROVE THE FREEZABILITY OF RAM SEMEN IN RECENT YEARS: a mini review
Sel'skokhozyaistvennaya Biologiya, 2017
One of the most important reasons why artificial insemination does not spread as much in cattle as sheep is that ram sperm is highly fragile against cryodamage and consequently the optimum fertility results can not be obtained from cervical inseminations compared to laparoscopic insemination. To establish an ideal ram sperm freezing model, many studies have been carried out for more than 60 years and various methods have been tested by making great efforts. One of the topics where these tests have been done intensively in recent years are additives used in freezing extenders in order to increase the tolerance of sperm against oxidative stress in frozen-thawed ram semen. In this review, covering the studies carried out between the years 2000-2016, we have mostly compiled the additives used in ram semen freezing media which have given better results compared to others supplements. It is understood that, usually, seminal plasma and proteins, thiol compounds, enzymatic antioxidants, sugars, fatty acids and vitamins have been studied as additive agents in recent years. As a result, this review suggests us that further studies are needed to explore the novel techniques and new additives, and their combinations should be tested in different doses in order to install an ideal cryopreservation template for ram semen. Furthermore, even though addition of various compounds to freezing media have improved freezability of ram semen in experimental conditions, anyway these results must be confirmed in field studies. Therefore, the value of sperm which potential fertility is predicted from laboratory survey must be compared with conception or lambing rates.