Cryopreservation of bison semen without exogenous protein in extender and its fertility potential in vitro and in vivo following fixed-time artificial insemination

2020 ◽  
Vol 152 ◽  
pp. 156-164
Author(s):  
Steve X. Yang ◽  
Gregg P. Adams ◽  
Jesus M. Palomino ◽  
Willian F. Huanca ◽  
Carl Lessard ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Fixed Time ◽  
Exogenous Protein ◽  
Fertility Potential

39 FERTILITY POTENTIAL OF FROZEN-THAWED WOOD BISON SEMEN USING EXTENDER WITHOUT EXOGENOUS PROTEIN

2016 ◽  
Vol 28 (2) ◽  
pp. 149
Author(s):  
S. X. Yang ◽  
G. P. Adams ◽  
J. M. Palomino ◽  
M. Anzar
Keyword(s):  
Test Group ◽  
Egg Yolk ◽  
Control Group ◽  
Computer Assisted ◽  
Exogenous Protein ◽  
Wood Bison ◽  
Fertility Potential ◽  
Bovine Oocytes

Cryopreservation of semen free of infectious agents is critical for the recovery and preservation of genetic diversity in Canada’s threatened wood bison populations. Egg yolk is a common constituent of conventional semen extenders, but it raises biosafety concerns related to transmission of infectious agents. The purpose of this experiment was to determine the fertility potential, both in vitro and in vivo, of semen frozen without the use of exogenous protein but with addition of cholesterol loaded cyclodextrin (CLC). Semen was collected by electro ejaculation from 4 wood bison bulls. Fresh sperm total motility and concentration were analysed using a computer-assisted sperm analyzer. Ejaculates with total sperm motility of >60% and a concentration of >200 × 106 sperm mL–1 were selected and pooled among bulls. For the control group, pooled semen was diluted to 50 × 106 sperm mL–1 with conventional extender containing 20% egg yolk and 7% glycerol. For the test group, pooled semen was diluted to 100 × 106 sperm mL–1 in Tris-citric acid buffer and incubated with 2 mg mL–1 CLC for 15 min before 1 : 1 dilution with 14% glycerol extender. Extended semen was placed in 0.5-mL straws, cooled, and frozen. Post-thaw motility analysis was conducted using a computer-assisted sperm analyzer, and the 2 collections with highest post-thaw motility were selected for fertility testing. Heterologous IVF was conducted using bovine oocytes obtained from an abattoir. After 20 to 22 h of in vitro maturation, the cumulus-oocyte complexes were placed into drops with either control or test semen. After 18 h, potential zygotes were denuded and moved to culture media. Cleavage and blastocyst rates were assessed on Day 4 and 8, respectively, from oocyte collection. The fertility potential of the semen was also tested in vivo using synchronized wood bison cows. At 24 h after hCG treatment, bison cows were assigned randomly to 2 groups and inseminated twice, 12 h apart, with frozen-thawed control semen (n = 23) or test semen (n = 23). Pregnancy was assessed 34 to 36 days after insemination by transrectal ultrasonography. Total sperm motility (mean ± standard error of the mean) of fresh semen was 80.2 ± 3.3%. The post-thaw motility was 41.7 ± 2.9% and 44.6 ± 3.3% for control and test semen, respectively. The cleavage rates of bovine oocytes fertilized in vitro with bison semen from the control group (n = 278) and test group (n = 299) were 49.9 ± 2.1% and 41.3 ± 4.5%, respectively, and the blastocyst rates were 17.7 ± 1.7% and 17.3 ± 2.5%, respectively. Pregnancy rates of wood bison artificially inseminated with control and test semen were 39 and 0%, respectively. In conclusion, wood bison semen frozen with CLC (without exogenous protein) resulted in post-thaw motility and in vitro fertility potential comparable to that of conventional egg yolk extender. However, CLC semen failed to fertilize in vivo, perhaps because of disruption of the processes of capacitation. The dichotomy between in vivo and in vitro results was surprising. It may be necessary to exercise caution when using IVF as a tool to assess fertility in vivo.

The Control Of Antithrombotic Prophylaxis And Therapy: A Pro-Coagulant Effect Of Heparin

1981 ◽  
Author(s):  
E Szwarcer ◽  
R Giuliani ◽  
E Martinez Aquino
Keyword(s):  
Blood Coagulation ◽  
Antithrombin Iii ◽  
Fixed Time ◽  
In Vitro Studies ◽  
In Vivo Studies ◽  
Clotting Factors ◽  
Platelet Poor Plasma ◽  
Normal Platelet

For studying heparin effect on blood coagulation and on inhibitors, the drug was added at increasing amounts to a normal platelet poor plasma (PPP), and to plasmas of patients with variable amounts of clotting factors (cirrhotic, pregnant, etc) -IN VITRO STUDIES-, and infused to the same individuals -IN VIVO STUDIES-. Modifications on two clotting assays (KCCT-TT) were compared to heparin potentiating effect on AntiXa (Denson & Bonnar tech).When studied IN VITRO, the sensibility of KCCT, TT, and AntiXa techniques for heparin measurement was similar. IN VIVO, an apparently greater sensibility using AntiXa technique was observed.For determining if this phenomena was related to a specific enhanced potentiating effect of the inhibitor against Xa, exerted by heparin IN VIVO, experiences were repeated IN VITRO and IN VIVO, measuring heparin effect on KCCT, TT, and on the inhibitor, studied against Xa and thrombin. A personal technique was used for the measurement of Antithrombin III heparin potentiating effect, using diluted platelet poor test plasma, heated (56°C 15’) and incubated with thrombin during a fixed time, and reading residual thrombin on citrated human PPP. IN VITRO, all techniques were similar in their ability to show heparin presence.IN VIVO, the potentiating effect of heparin on the inhibitor, measured against Xa or thrombin, was greater than the changes obtained on KCCT or TT.So, AntiXa-Antithrombin III techniques seem to be more sensitive for heparin measurement IN VIVO.This “dissociation” of results in between the potentiating effect on the inhibitor, that is not simultaneously exerted on global coagulation, is interpreted as a heparin pro-coagulant effect, exerted by the drug IN VIVO.

Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen

2008 ◽  
Vol 69 (4) ◽  
pp. 513-522 ◽  
Author(s):  
C.M. O’ Meara ◽  
J.P. Hanrahan ◽  
N.S. Prathalingam ◽  
J.S. Owen ◽  
A. Donovan ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Functional Tests

Artificial insemination technology for ratites: a review

2008 ◽  
Vol 48 (10) ◽  
pp. 1284 ◽  
Author(s):  
I. A. Malecki ◽  
P. K. Rybnik ◽  
G. B. Martin
Keyword(s):  
Artificial Insemination ◽  
Genetic Improvement ◽  
Quantitative Methods ◽  
Practical Method ◽  
Prolonged Storage ◽  
Female Ratio ◽  
Semen Collection ◽  
Semen Storage

In ratite farming, the low male to female ratio in the mating system restricts genetic improvement and prevents reduction of the number of males kept on-farm for fertilisation of the female flock. These issues can be overcome and the industry can better realise its potential by using artificial insemination (AI) technology. It is the only practical method for intensive genetic improvement of reproduction and the production of eggs, chicks, oil, meat and leather. For AI to be feasible, we need reliable methods for semen collection, artificial insemination, prolonged storage of spermatozoa in the female tract, high rates of lay, efficient protocols for semen storage, and a panel of quantitative methods for measuring true fertility and hatchability, sperm supply rates in vivo and sperm viability in vitro. For both emus and ostriches, prolonged sperm storage in females has already been demonstrated. Methods for semen collection and artificial insemination, using animal-friendly techniques, have also been developed. Semen storage and cryopreservation protocols are yet to be optimised and we still need to overcome the male-dependent rate of lay, but adoption of AI technology by the ratite industries is now feasible. It also seems likely that these technologies will be relevant to wild ratites that need intensive conservation efforts, such as cassowaries, rheas and ostrich subspecies.

233 HIGH NUMBERS OF ANTRAL FOLLICLES INFLUENCE THE IN VITRO EMBRYO PRODUCTION, BUT NOT THE CONCEPTION RATE OF FIXED-TIME ARTIFICIAL INSEMINATION IN NELORE CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 206
Author(s):  
G. M. G. Santos ◽  
K. C. Silva-Santos ◽  
T. R. R. Barreiros ◽  
F. Morotti ◽  
B. V. Sanches ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Fixed Time ◽  
Device Removal ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Antral Follicles ◽  
Conception Rates ◽  
In Vitro Embryo Production ◽  
Embryo Grade

The aim of this study was to compare the conception rates to fixed-time artificial insemination (FTAI) and in vitro embryo production between Nelore cows with high or low antral follicle counts (AFC). First, multiparous Nelore cows (Bos indicus, n = 547, 40–60 days postpartum) were subjected to synchronization of ovulation. Randomly during their oestrous cycle (Day 0), cows received an intravaginal device containing 1.9 g of P4 (CIDR®) and 2 mg of oestradiol benzoate (Estrogin®), intramuscularly. At device removal (Day 8), cows received 500 µg of PGF2α (Ciosin®), 300 IU of eCG (Novormon®), and 1 mg of oestradiol cipionate (ECP®), intramusculary. All cows were inseminated 48 h after P4 device removal. Antral follicles = 3 mm were counted using an intravaginal microconvex transducer (Day 0), and cows were assigned to groups of high (G-High, = 25 follicles, n = 183), intermediate (G-Intermediate, 16–20 follicles, n = 183), or low AFC (G-Low, = 10 follicles, n = 181). In another study to compared the in vitro embryo production, Nelore cows (n = 66, 72–96 months) were subjected to ultrasound-guided follicular aspiration using an intravaginal microconvex array transducer (7.5 MHz). The COC were selected and cows were assigned to groups according to the oocyte production: G-High (n = 22, = 40 oocytes), G-Intermediate (n = 25, 18–25 oocytes), or G-Low (n = 19, = 7 oocytes). Previously tested semen from a single bull was used for IVF using a previously described protocol (Silva-Santos et al. 2014 Reprod. Domest. Anim. 49, 228–232). The oocyte and embryo production (viable embryo: grade I, II, III; vitrifiable embryo: grade I, II) were evaluated. The number of follicles was evaluated by Kruskal-Wallis, and the chi-square test was used for data on oocyte and embryo production (P = 0.05). The average follicular population was 30.7 ± 5.7 (G-High), 18.6 ± 1.64 (G-Intermediate), and 7.8 ± 2.4 follicles (G-Low; P < 0.05), but there were no differences in the conception rates among groups (51.9 v. 48.6 v. 58.6%, respectively; P > 0.05). The total number of oocytes recovered were 1109 (G-High), 534 (G-Intermediate), and 101 (G-Low; P < 0.05). The mean number of viable oocytes was 40.4 ± 10.6 (G-High), 14.8 ± 3.0 (G-Intermediate), and 3.8 ± 1.1 (G-Low; P < 0.05) and the percentage of viable oocytes was 80% (G-High), 69% (371/534, G-Intermediate), and 71% (G-Low; P < 0.05). Cleavage rate was 79% (G-High), 74% (348/472, G-Intermediate), and 71% (G-Low; P < 0.05), and blastocyst rate was 42% (G-High), 32% (153/472, G-Intermediate), and 13% (G-Low; P < 0.05). The number of viable embryos was 18.4 ± 6.7 (G-High), 6.1 ± 3.6 (G-Intermediate), and 0.6 ± 0.7 (G-Low; P < 0.05) and the percentage of vitrifiable embryos was 81% (G-High), 77% (118/153, G-Intermediate), and 58% (G-Low; P < 0.05). Therefore, Nelore cows with high oocyte production had ~10-fold higher oocyte production and produced ~30-fold more embryos compared with the low AFC group. In conclusion, AFC had no influence on the conception rates to FTAI; however, Nelore cows with high oocyte production exhibited higher in vitro embryo production.

Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects

1999 ◽  
Vol 11 (1) ◽  
pp. 1 ◽  
Author(s):  
D. P. Evenson
Keyword(s):  
Chromatin Structure ◽  
Male Fertility ◽  
Nuclear Dna ◽  
Early Embryo ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay ◽  
Fertility Potential ◽  
Post Stress

An important goal of modern analyses of semen is to elucidate the molecular traits of mammalian sperm chromatin structural abnormalities, defined here as ‘uncompensable’, that lead to abnormalities in fertility, pronuclear formation, early embryo quality and pregnancy outcome. Sperm with uncompensable nuclear abnormalities are able to fertilize oocytes both in vivo and in vitro; however, due to the uncompensable trait(s), the embryo development may be abnormal. Uncompensable nuclear traits can be experimentally induced in bull sperm by a mild thermal insult to the testis. Sperm nuclear morphology abnormalities seen in ejaculates 11-days post stress are likely related to molecular changes in chromatin observed 3-days post stress by the flow cytometric sperm chromatin structure assay (SCSA). The SCSA measures the susceptibility of sperm nuclear DNA to denaturation in situ. This susceptibility has been correlated with the presence of DNA strand breaks that may be derived in part by oxidative stress and possibly by a unique, abortive apoptotic mechanism. The extent of DNA denaturation is not significantly related to the level of disulfide bonding between the chromatin protamines. The use of human sperm with uncompensable nuclear traits for artificial reproductive techniques is also discussed. The goal of this research is to remove from semen doses those sperm with uncompensable nuclear traits and thereby increase male fertility potential. Extra key words: male fertility potential, sperm chromatin structure assay (SCSA).

scholarly journals Use of Bayesian Inference to CorrelateIn VitroEmbryo Production andIn VivoFertility in Zebu Bulls

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mateus José Sudano ◽  
André Maciel Crespilho ◽  
Claudia Barbosa Fernandes ◽  
Alicio Martins Junior ◽  
Frederico Ozanam Papa ◽  
...  
Keyword(s):  
Bayesian Inference ◽  
Fixed Time ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Conception Rates ◽  
Using Data ◽  
Pronuclear Formation ◽  
Combined Data

The objective of this experiment was to testin vitroembryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilizedin vitrousing sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimatein vivobull fertility using data from IVP.

21 THE INJECTION OF CORTISOL TO UTERUS INCREASES THE IMPLANTATION RATE AND LITTER SIZE IN PIG ARTIFICIAL INSEMINATION USING CYROPRESERVED SPERMATOZOA

2011 ◽  
Vol 23 (1) ◽  
pp. 117
Author(s):  
M. Shimada ◽  
T. Okazaki
Keyword(s):  
Litter Size ◽  
Seminal Plasma ◽  
Artificial Insemination ◽  
Implantation Rate ◽  
Fertilization Rate ◽  
Dependent Manner ◽  
Vitro Fertilization ◽  
Novel Method

Cryopreserved boar spermatozoa are not routinely available to swine artificial insemination (AI) because conception and farrowing rates, along with litter size, have remained low. We have reported the positive roles of seminal plasma in frozen–thawed sperm functions (Okazaki et al. 2009 Theriogenology 71, 491–498). Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the implantation rate. Thus, the factors in seminal plasma act not only on sperm but also on uterus to induce successful fertilization and implantation in pig AI using cryopreserved spermatozoa. To test this hypothesis, we identified the factors in seminal plasma and then developed novel pig AI method using cryopreserved spermatozoa. The sperm-rich fraction was collected weekly from each boar using the gloved-hand technique. The seminal plasma was removed just after collection by centrifuge and then was frozen as described in our previous study (Okazaki et al. 2009 Theriogenology 71, 491–498). When the frozen–thawed sperm was incubated with Fluo-3/AM to determine the level of intercellular Ca2+, the level of Ca2+ was increased in a time-dependent manner, and spontaneous capacitation that was judged by tyrosine phosphorylation of sperm protein by Western blotting (Shimada et al. 2008 Development 135, 2001–2011), was also induced in post-thawed sperm. The addition of EGTA to thawing solution significantly suppressed the Ca2+-induced capacitation. Moreover, the treatment increased fertilization rate in in vitro fertilization and in vivo in artificial insemination as similar as those in sperm with seminal plasma. The same number of blastocyst was collected from uterus by AI using post-thawed sperm with EGTA. However, the pregnancy rate remained low, and the number of leukocytes in the uterus was increased. In the next experiment, we examined in seminal plasma, the level of cortisol that has been known to play an important role in controlling immune function. The results showed that cortisol (1.0 ng mL–1) was detected in seminal plasma. When the sows of natural oestrus were twice artificial inseminated with or without cortisol, the injection of cortisol (5 μg/50 mL) to uterus with sperm significantly decreased the number of leukocytes in the uterus or endometrium at 24 to 36 h after AI. The low number of leukocytes in the uterus was similar to that in uterus injected fresh semen. The cortisol injection significantly increased the implantation rate and litter size of sows as compared to AI without cortisol (implantation rate; 83% v. 51%, litter size; 10.6 v. 7.3). From these results, we concluded that the injection of cortisol with frozen–thawed spermatozoa by EGTA-containing solution was a novel method of pig AI using cryopreserved spermatozoa. This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).

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