Fertility analysis of bovine semen by in vitro fertilization (original) (raw)
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Annals of Tropical Medicine and Public Health, 2019
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2017
In vitro embryo production is a well-established and commercial technique, being nowadays the best choice for embryo production in some breeds. Therefore, better results can be achieved with the selection of high in vitro fertility bulls. Understanding why a high fertility bull produce more blastocysts than a low fertility one is the key for bull selection. Since embryo kinetics can be linked with embryo viability (Edwards, 2003), it could be a powerful tool to identify differences between these two groups of bulls. The aim of this study was to evaluate early embryo kinetics and fertilization rate from bulls with high (HF) and low (LF) in vitro fertility. For bull selection, a commercial laboratory database was assessed. Bulls were ranked based on blastocyst/cleavage rate (embryo development rate). Ten bulls with high (n=5) and low (n=5) in vitro fertility were used for 5 manipulation of in vitro embryo production, as described by Pontes et al. (2010). Ten oocytes from each manipulation (n=50) were stained with 1 mg/ml Hoechst 33342 (Sigma), washed, mounted on microscope slides and examined with epifluorescence microscopy (Olympus IX80, Olympus Corporation, Tokyo, Japan). The presumptive zygotes were classified in three categories: negative fertilization (1 PN); normal fertilization (2 PN), and polyspermy (>2 PN). Embryos were classified by their specific stage of development (2; 3-4; 6 or 8 cell stage) at 24, 36, 48, 60, 72 hpi (hours postinsemination). Cleavage rate was assessed at day 3 (D3) of embryo culture, viable blastocysts and embryo development rates were assessed at day 7 (D7). Data were analyzed using PROC GLIMMIX of SAS (SAS ® 9.3 Institute Inc., Cary, NC, USA, 2003). Blastocyst rate was higher in the HF group (29.4%) than in the LF (16.0%-P<0.0001), similarly to embryo development rate (HF = 34.0%; HL = 189%; P<0.0001). There was no significant difference in cleavage rate (HF=86.7%; LF= 84.9%; P= 0.2581), neither in embryo kinetics, in all of the evaluated periods (P>0.05). No difference was found in negative fertilization (HF=10.69%; LF= 8.80%; P=0.9925), nor in polyspermy between groups (HF=16.18%; LF=29.20%; P=0.6066). However, normal fertilization was higher in the HF group (72.0%) than in LF group (62.0%) (P=0.0332). In conclusion, early embryo kinetics could not explain the difference in blastocyst rate between high fertility (HF) and low fertility (LF) bulls. Nevertheless, HF bulls had more normal fertilization than LF bulls.
Livestock Research for Rural Development
The present investigation was aimed at evaluating the bulls and their semen from production to insemination in a routine artificial insemination (AI) programme in Bangladesh. Five crossbred bulls were examined for breeding soundness and their semen was preserved and evaluated with respect to sperm motility, and proportion of spermatozoa with normal acrosome, midpiece and tail. Semen was sampled immediately after collection, pre-diluted, cooling down to +4 0 C, and storage at -196 0 C for 1 day, 7 days and 3 months in Central Cattle Breeding Station (CCBS). Semen was transported from CCBS to District AI centre at Mymensingh, and then samples were collected after 7 days, 3 months and immediately before insemination. From the District AI centre, semen was transported to the Sub-centre, Fulbaria and sampled there within 7 days after transportation and immediately before insemination.
In vitro assessment of sperm from bulls of high and low field fertility
Theriogenology, 2011
The aim of this study was to investigate the reasons for differences in field fertility of bulls following insemination with frozen-thawed semen. The study was carried out in two separate parts over two years and comparisons were made between 5 high and 4 low fertility Holstein Friesian bulls as determined by their either 90 day non-return rate (Year 1) or calving rate (Year 2). Two high fertility Limousin bulls were included in Year 1 for comparative purposes. The ability of sperm from each bull to penetrate artificial mucus was assessed (Year 1 = 7 replicates; Year 2 = 5 replicates). Glass capillary tubes (2 per bull per replicate) were filled with artificial mucus and incubated with sperm stained in 1% Hoechst 33342 for 30 min at 37 o C. The number of sperm were subsequently counted at 10 mm intervals along the tube between 40 and 80 mm markers. Sperm mitochondrial activity of each bull was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay (4 replicates in each year). Sperm were incubated with MTT for 1 h at 37 o C following which the absorbance of formazan was read using a spectrophotometer. Sperm viability after thawing was assessed for each bull using a live/dead sperm viability kit (Year 1 = 3 replicates; Year 2 = 4 replicates). A minimum of 250 cells were assessed per bull in each replicate and classified as either live or dead. Finally, the ability of sperm to fertilise oocytes in vitro and their ability to develop to blastocyst stage embryos were assessed (5 replicates in each year involving 220 to 306 oocytes per bull). Data transformation to normalise residuals was required for mucus sperm penetration (square root) and IVF (cleavage and blastocyst rate) results (arcsin). The mean number of sperm counted at each 10 mm mark between 40 and 80 mm was higher in the high fertility (56.0; 95%CI 39.5 to 75.3) compared to the low fertility 3 (42.9; 95% CI 29.3 to 59.1) Holstein Friesian bulls but the difference did not reach formal significance (P = 0.09). Fertility status had no effect on the ability of sperm to reduce MTT to formazan (mean absorbance 0.34 ± 0.051 and 0.30 ± 0.044) or on the percentage of live sperm per straw (mean 47.3 ± 5.47 and 32.4 ± 4.66) for high and low fertility Holstein Friesian bulls respectively. Oocyte cleavage rate following insemination with sperm from high fertility Holstein Friesian bulls was significantly higher than with sperm from low fertility Holstein Friesian bulls [76.7% (95%CI 60.9 to 89.4) and 55.3 (95%CI 40.4 to 69.7) respectively, P = 0.04]. There was no significant effect of bull fertility on blastocyst rate [34.7% (95%CI 21.1 to 49.6) and 24.2 % (95%CI 14.1 to 36.0) for the high and low fertility Holstein Friesian bulls, respectively; P = 0.2]. In conclusion, sperm from high fertility bulls tended to be more effective in penetrating artificial mucus and to have an increased ability to fertilise oocytes in vitro; however, once fertilisation occurred subsequent embryo development was not significantly affected by fertility status.
Evaluation of bull performance based on in vitro fertilization studies
1997
The first part of this study investigated if differences among bulls as semen donors, and sperm concentrations affected cleavage rate of oocytes. Three levels of sperm concentration and their effect on cleaving oocytes were analyzed. Frozen-thawed semen samples from five Holstein bulls and oocytes (n=728) harvested from slaughterhouse ovaries were used in this experiment. Oocytes were matured in vitro with Hams' F-10 and estrus cow serum (ECS) media, then fertilized in vitro using Tyrode Albumin Lactate Pyruvate (TALP) media. The resulting embryos were subsequently cultured using Hams' F-10 and fetal calf serum (FCS) in the presence of oviductal cells . The three sperm concentrations used in the study were 2xl06, lxlO 6 and 0.5xl06 /ml. Approximately 5672 h after addition of sperm, cleavage as indicated by a 2-cell stage embryo was observed and recorded. A significant difference (P< 0.001) in cleavage rate was observed among the five bulls. Sperm concentration did not aff...
Livestock Science, 2012
In order to provide information that may help researchers to understand the main cause(s) of differences in bull fertility frequently observed in field trials, this study aimed to investigate conception rates as well as several in vitro sperm characteristics of different sires of unknown fertility utilized in a Timed-AI (TAI) program. Suckled Nelore cows submitted to the same TAI protocol were allocated into eight breeding groups of approximately 120 animals each. Frozen semen doses from three Angus bulls and three different batches from each bull were utilized. Approximately 100 doses from each batch were used in TAI. Sires, batches and AI technicians were equally distributed across breeding groups. Cows were examined for pregnancy diagnosis 40 d after TAI. For in vitro sperm analyses, the same thawing procedure was repeated in the laboratory to mimic field conditions. The following in vitro sperm characteristics were assessed: computerized motility, thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, morphology, morphometry and chromatin structure. No effect of breeding group, body condition score, AI technician and sire was observed. However, some significant differences among bulls were detected in laboratory analyses. Semen from sire presenting numerically lower (P > 0.05) pregnancy/AI also presented lower (P b 0.05) values in all sperm characteristics analyzed in thermal resistance test at 4 h (Total Motility, Progressive Motility, Average Path Velocity, Straight-Line Velocity, Curvilinear Velocity, Amplitude of Lateral Head Displacement, Beat Cross Frequency, Straightness, Linearity, and Percentage of Rapidly Moving Cells), higher (P b 0.05) Major and Total Defects in sperm morphological test, lower (P b 0.05) Length, Ellipticity and Fourier parameter (Fourier 0) in sperm morphometric analysis as well as higher (P b 0.05) chromatin heterogeneity. It was concluded that, although no bull effect was observed in the field experiment, the sire that presented numerically lower pregnancy/AI also presented lower semen quality according to the laboratory analyses performed.
Conception Rates of Holstein-Friesian Cows Inseminated Artificially with Reducing Frozen Semen Doses
Media Peternakan, 2016
Conception rates of Holstein Friesian (HF) acceptors were evaluated in artificial insemination (AI) mating using three frozen semen doses of the straws per volume of 0.25 cc. Frozen semen consisted of a standard dose by 25 x 10 6 sperms (L.1) and two reduced doses by 20 x 10 6 sperms (L.2) and 15x10 6 sperms (L.3). This field study was conducted in dairy cattle area of Lembang NBMCU, from August 2011 to March 2012. Frozen semens were obtained from two HF national progeny tested young bulls. Conception was detected by rectal palpation, around 60-90 days after AI mating. Conception rates (CR %) were calculated as a ratio between pregnant acceptors to the total AI mated cows. Some general linear model analyzes were conducted by considering fixed variables of semen dose (3), bull (2), inseminator (6) and parity (5). The results showed that three frozen semen doses did not significantly affect (P>0.05) on CR (%). By decreasing semen dose to L.3 gave CR (%) similar to L2 even to L1. It was concluded that frozen semen doses of HF bulls at the levels of 20 million to 15 million sperms per straw per volume of 0.25 cc resulted CR (%) similar to the standard one.