Bovine sperm selection procedure prior to cryopreservation for improvement of post-thawed semen quality and fertility

Abstract Background The application of cryopreservation and artificial insemination technology have contributed to the advancement of animal reproduction. However, a substantial proportion of spermatozoa undergoes alterations and loses their fertility during cryopreservation, rendering the frozen-thawed semen impractical for routine use. Cryopreservation is known to reduce sperm lifespan and fertility. Variation in cryosurvival of spermatozoa from different sires and even with the individual sire is common in artificial insemination (AI) centers. Our goal is to improve post-thawed semen quality by optimization of cryopreservation technique through sperm selection prior to cryopreservation process. Results Our strategy of sperm selection based on rheotaxis and thermotaxis (SSRT) on macrosale in a rotating fluid flow demonstrated the ability to maintain the original pre-freezing structural integrity, viability and biological function related to fertilization competence. This strategy has a positive effect on the cryosurvival and fertilizing abilities of spermatozoa as supported by the improvement on pregnancy rate of Japanese Black heifers and Holstein repeat breeders. This technique protected further sublethal damage to bovine spermatozoa (higher % cryosurvival than the control) and resulted in the improvement of DNA integrity. Prefreeze selected spermatozoa demonstrated slower and controlled capacitation than unprocessed control which is thought to be related to sperm longevity and consequently to appropriate timing during in vivo fertilization. Conclusions These results provide solid evidence that improvement of post-thawed semen quality by SSRT method is beneficial in terms of cryosurvival, longevity of post-thawed sperm, and optimization of in vivo fertilization, embryo development and calving as supported by the favorable results of field fertility study.

Download Full-text

A comparative study of boar semen extenders with different proposed preservation times and their effect on semen quality and fertility

Acta Veterinaria Brno ◽

10.2754/avb201685010023 ◽

2016 ◽

Vol 85 (1) ◽

pp. 23-31 ◽

Cited By ~ 3

Author(s):

Marina Anastasia Karageorgiou ◽

Georgios Tsousis ◽

Constantin M. Boscos ◽

Eleni D. Tzika ◽

Panagiotis D. Tassis ◽

...

Keyword(s):

Artificial Insemination ◽

Semen Quality ◽

Medium Term ◽

Semen Collection ◽

Chromatin Integrity ◽

Boar Semen ◽

Short Time

The present study compared the quality characteristics of boar semen diluted with three extenders of different proposed preservation times (short-term, medium-term and long-term). A part of extended semen was used for artificial insemination on the farm (30 sows/extender), while the remaining part was stored for three days (16–18 °C). Stored and used semen was also laboratory assessed at insemination time, on days 1 and 2 after the collection (day 0). The long-term extender was used for a short time, within 2 days from semen collection, with the aim to investigate a possible advantage over the others regarding laboratory or farm fertility indicators at the beginning of the preservation time. Viability, motility, kinetic indicators, morphology and DNA fragmentation were estimated. The results showed reduced viability, higher values for most of the kinetics, and higher immotile spermatozoa from day 1 to day 2 in all extenders; however, the long-term extender was superior compared to the other two on both days. With regard to morphology and chromatin integrity, the percentage of abnormal and fragmented spermatozoa increased on day 2 compared to day 1 for all of the extenders. However, based on the farrowing rate and the number of piglets born alive after the application of conventional artificial insemination within 2 days from semen collection/dilution, it was found that the medium-term diluents were more effective. In conclusion, it seems that the in vivo fertilization process involves more factors than simply the quality of laboratory evaluated sperm indicators, warranting further research.

Download Full-text

A Review on Animals Semen Characteristics: Fertility, Reproduction and Development

Asian Journal of Advances in Agricultural Research ◽

10.9734/ajaar/2019/v10i230024 ◽

2019 ◽

pp. 1-9

Author(s):

Farzad Moradpour

Keyword(s):

Structural Integrity ◽

Current Knowledge ◽

Semen Analysis ◽

Kinetic Property ◽

Genetic Material ◽

Computer Assisted ◽

Semen Characteristics ◽

The Individual

In this research, the goal of review was summarizing the current knowledge of the methods available to assess in vitro quality of frozen-thawed bovine spermatozoa also, a review on animal’s semen characteristics: fertility, reproduction and development after AI with that semen. Artificial insemination (AI) is the first generation reproductive biotechnology that has made a deep contribution to the genetics improvement in several animals. A fertile ejaculate must meet certain semen characteristics quality standards, such as: normal morphology, active energy metabolism, progressive motility, structural integrity and functionality of the membrane, penetration capacity and optimum transfer of genetic material. The percentage of total motile spermatozoa in normal canine ejaculates is between 70 to 90%. By the way, there are a lot of parameters that able to change on the composition and structure of various sperm plasma member domains, such as change temperature and sensitive to any theirs environments in vivo and vitro (tropical climates), season also nutrition. Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements that gives extensive information about the kinetic property of the ejaculate based on measurements of the individual sperm cells.

Download Full-text

Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Animals ◽

10.3390/ani9060304 ◽

2019 ◽

Vol 9 (6) ◽

pp. 304 ◽

Cited By ~ 1

Author(s):

Giusy Rusco ◽

Michele Di Iorio ◽

Pier Paolo Gibertoni ◽

Stefano Esposito ◽

Maurizio Penserini ◽

...

Keyword(s):

Brown Trout ◽

Semen Quality ◽

Sperm Quality ◽

Egg Yolk ◽

Dna Integrity ◽

Experimental Conditions ◽

C 30 ◽

Thawing Rate

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

Download Full-text

Hydroxy-selenomethionine as an organic source of selenium in the diet improves boar reproductive performance in artificial insemination programs

Journal of Animal Science ◽

10.1093/jas/skab320 ◽

2021 ◽

Author(s):

Ana Paula P Pavaneli ◽

Cristian H G Martinez ◽

Denis H Nakasone ◽

Ana Carolina Pedrosa ◽

Maitê V Mendonça ◽

...

Keyword(s):

Seminal Plasma ◽

Reproductive Performance ◽

Artificial Insemination ◽

Semen Quality ◽

Sperm Quality ◽

Sperm Count ◽

Dietary Treatments ◽

Gpx Activity ◽

Organic Source

Abstract This study aimed to compare different selenium (Se) sources in the diet on boar's semen quality and fertility. For this, 28 boars aged 8 to 28 months were fed with the following dietary treatments for 95 days: 0.3 mg Se/kg as sodium selenite (SS, n = 14) and 0.3 mg Se/kg as hydroxy-selenomethionine (OH-SeMet, n = 14). During this period, two experiments were carried out. In experiment 1, the semen of all boars was evaluated every 2 weeks. Raw semen was initially evaluated for the processing of seminal doses, which were stored at 17 °C for 72 h, followed by sperm quality assessments. Furthermore, Se concentration and glutathione peroxidase (GPx) activity were measured in the seminal plasma. In experiment 2, 728 females were inseminated weekly with seminal doses from boars of the different experimental groups to further assess in vivo fertility and litter characteristics. Results demonstrated that boars fed OH-SeMet had more Se in their seminal plasma (p < 0.05), showing the greater bioavailability of the organic source in the male reproductive system. Moreover, boars fed OH-SeMet tended (p < 0.10) towards a higher total sperm count in the ejaculate (66.60 vs. 56.57 × 10 9 sperm), and the number of seminal doses (22.11 vs. 18.86; 3 × 10 9 sperm/dose) when compared to those fed SS. No effect of the dietary treatments was observed on GPx activity in seminal plasma (p > 0.05), as well as on raw and stored semen quality (p > 0.05). Under in vivo conditions, seminal doses from boars fed OH-SeMet tended (p < 0.10) towards a higher pregnancy rate at weeks 3, 5, and 8, and also resulted in a higher (p < 0.05) percentage of pregnant females in the overall period (99.30 vs. 97.00). In conclusion, the replacement of SS with OH-SeMet in boars' diet can improve sperm production and results in better reproductive performance for them, bringing greater productivity and profitability to artificial insemination centers and commercial pig farms.

Download Full-text

The Role of Selected Natural Biomolecules in Sperm Production and Functionality

Molecules ◽

10.3390/molecules26175196 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5196

Author(s):

Eva Tvrdá ◽

Filip Benko ◽

Tomáš Slanina ◽

Stefan S. du Plessis

Keyword(s):

Molecular Mechanisms ◽

Structural Integrity ◽

Biological Effects ◽

Natural Compounds ◽

In Vivo Studies ◽

Dna Integrity ◽

Sperm Production ◽

Reproductive Dysfunction

Emerging evidence from in vivo as well as in vitro studies indicates that natural biomolecules may play important roles in the prevention or management of a wide array of chronic diseases. Furthermore, the use of natural compounds in the treatment of male sub- or infertility has been proposed as a potential alternative to conventional therapeutic options. As such, we aimed to evaluate the effects of selected natural biomolecules on the sperm production, structural integrity, and functional activity. At the same time, we reviewed their possible beneficial or adverse effects on male reproductive health. Using relevant keywords, a literature search was performed to collect currently available information regarding molecular mechanisms by which selected natural biomolecules exhibit their biological effects in the context of male reproductive dysfunction. Evidence gathered from clinical trials, in vitro experiments and in vivo studies suggest that the selected natural compounds affect key targets related to sperm mitochondrial metabolism and motion behavior, oxidative stress, inflammation, DNA integrity and cell death. The majority of reports emphasize on ameliorative, stimulating and protective effects of natural biomolecules on the sperm function. Nevertheless, possible adverse and toxic behavior of natural compounds has been indicated as well, pointing out to a possible dose-dependent impact of natural biomolecules on the sperm survival and functionality. As such, further research leading to a deeper understanding of the beneficial or adverse roles of natural compounds is necessary before these can be employed for the management of male reproductive dysfunction.

Download Full-text

P–045 A retrospective study on Hyaluronic acid binding sperm selection in ICSI cycles

Human Reproduction ◽

10.1093/humrep/deab130.044 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

J Y Lo ◽

S T Tee

Keyword(s):

Pregnancy Rate ◽

Fertilization Rate ◽

Mature Sperm ◽

Utilization Rate ◽

Dna Integrity ◽

Sperm Selection ◽

Miscarriage Rate ◽

Icsi Cycle ◽

Significant Difference

Abstract Study question Does Hyaluronic acid(HA) binding sperm selection prior ICSI produce better outcome compare to PVP? Summary answer No significant difference in fertilization rate, blastocyst utilization rate, pregnancy rate and miscarriage rate in HA vs PVP group. What is known already HA is natural, non-sulphated glycosaminoglycan secretion that abundantly found in the cervical mucus and the cumulus oophorus complex (COC). In in-vivo, HA binding sites on the sperm plasma membrane indicate sperm maturity, mature sperm reaching HA-coated COC can bind and digest HA and subsequent hyperactivation further facilitate fertilization. In IVF, the use of HA tries to mimic in-vivo to select mature sperm with high DNA integrity prior to ICSI. In HA, movement of mature sperm is ‘slowed’ thus allow the selection of sperm to be used in ICSI. Sperm immaturity is known to associated with aneuploidy incidence in the embryos. Study design, size, duration ICSI cycle using HA (N = 83) was adapted from January to December 2020 while ICSI cycle using PVP (N = 133) was adapted from January 2018 to December 2019. Mean age of patient were 35.64±4.33 vs 34.15±4.75 for HA vs PVP group respectively. Fertilization rate, blastocyst utilization rate, pregnancy rate and miscarriage rate were recorded. This study included all ICSI cycle and both frozen and fresh embryo transfer data. Surgical retrieved sperm was excluded from this study. Participants/materials, setting, methods A 1.5ul of treated spermatozoa suspension was connected with a pipette tip to a 5ul droplet of fresh holding medium (Cook Gamete Buffer). Simultaneously, a 5ul droplet of HA medium (Origio SpermSlow) was connected to the 5ulL droplet of holding medium in a ICSI dish (Sparmed Oosafe) covered with oil (Vitrolife Ovoil). Sperm which were ‘slowed’ in the Sperm slow droplets with normal morphology according to WHO 2010 guideline were selected for ICSI at 400X. Main results and the role of chance The fertilization rate of the HA vs PVP- binding sperm are 68.6% vs 66.2%. As for the blastocyst utilisation rate is 61.6% vs 73.22% for HA vs PVP- binding sperm group. Pregnancy and miscarriage rate for HA vs PVP are 42.3% vs 51.5% and 19.4% vs 26.2% respectively. There was no significant difference in the fertilization rates, blastocysts utilisation rate, pregnancy rate and miscarriage rate between the HA vs PVP- binding sperm groups (P < 0.05). However, a trend of higher pregnancy (51.5% vs 42.3%; P = 0.279) and miscarriage rate were observed in the PVP group (26% vs 19%; P = 0.545) as compared to the HA group, but the difference was not statistically significant. The reason behind this might be the HA assist to select the mature sperm with higher DNA integrity and low frequency of chromosomal aneuploidies which contribute to the lower miscarriage rate in the study. Limitations, reasons for caution This is a retrospective study on HA binding sperm selection vs PVP prior to ICSI. Further research which includes a large number of RCT sample size should be warranted. Wider implications of the findings: The HA- sperm binding selection ICSI might only be beneficial to certain group of patients (high- DNA fragmentation sperm). A larger RCT study may be necessary to establish a relationship between HA-sperm binding selection vs aneuploidy rate via PGT analysis. Trial registration number Not applicable

Download Full-text

Improving the Rabbit Semen Cryopreservation Protocol: Comparison Between Two Extenders and Inseminating Doses

Annals of Animal Science ◽

10.2478/aoas-2020-0023 ◽

2020 ◽

Vol 20 (3) ◽

pp. 887-898

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Maria Antonietta Colonna ◽

Michele Schiavitto ◽

Maria Silvia D’Andrea ◽

...

Keyword(s):

Liquid Nitrogen ◽

Sperm Motility ◽

Artificial Insemination ◽

Sperm Quality ◽

Dna Integrity ◽

Semen Cryopreservation ◽

Fertilizing Ability ◽

Cryopreservation Protocol

AbstractThis study has been designed to optimize the semen freezing protocol in rabbits, in this regard we compared a Tris-citrate-glucose (TCG) extender with a commercial one (Cortalap®), that to the best of our knowledge has never been used up to now on the in vitro freezability and fertilizing ability of cryopreserved rabbit semen. Two different inseminating semen doses were considered. Five pooled semen samples were divided into two subsamples and each of them were diluted to a ratio 1:1 (v:v) with a freezing extender composed of TCG or Cortalap® containing 16% of dimethylsulfoxide and 0.1 mol/L of sucrose. The extended semen was filled into 0.25 mL plastic straws and frozen above a liquid nitrogen surface. After thawing (50°C/10 seconds) we determined sperm motility, viability, membrane functionality, acrosome and DNA integrity. Our results showed that the Cortalap® extender significantly improved the in vitro post-thaw sperm quality, in comparison to TCG. When we compared the extenders in vivo, no significant differences in the reproductive performances were observed independently by inseminating doses used. In this study we demonstrated that Cortalap® extender can be used as an alternative to TCG. Thus, the Cortalap® being a ready to use extender, implies a reduction of time, mistakes and microbial contaminations during its preparation. This discovery results as significant because it provides beyond an important contribution to the creation of the first Italian semen cryobank of rabbit breeds and also for livestock rabbit farms based on artificial insemination (AI) program.

Download Full-text

Coenzyme Q10 ameliorates cadmium induced reproductive toxicity in male rats

Physiological Research ◽

10.33549/physiolres.934000 ◽

2019 ◽

pp. 141-145 ◽

Cited By ~ 5

Author(s):

R. Saha ◽

S. Roychoudhury ◽

K. Kar ◽

A.C. Varghese ◽

P. Nandi ◽

...

Keyword(s):

Semen Quality ◽

Reproductive Toxicity ◽

Sperm Concentration ◽

Antioxidant Defense System ◽

Protective Role ◽

Male Rats ◽

Dna Integrity ◽

Water Control ◽

Progressive Motility

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.

Download Full-text

Effects of the Bull on Conception Rate of Dairy Cows in Different Seasons and According to AI Type

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.82554 ◽

2018 ◽

Vol 46 (1) ◽

pp. 6

Author(s):

Gabriela Bueno Luz ◽

Andressa Stein Maffi ◽

Lucas Balinhas Farias ◽

Eduardo Goulart Xavier ◽

Márcio Erpen Lima ◽

...

Keyword(s):

Reproductive Performance ◽

Artificial Insemination ◽

Semen Quality ◽

Dairy Farm ◽

Conception Rate ◽

Reproductive Efficiency ◽

Factors Affecting ◽

Holstein Breed ◽

The Individual ◽

Animal Category

Background: Reproductive efficiency is one of the most important factors affecting the performance dairy cattle, and fixedtime artificial insemination (TAI) is an important biotechnology factor employed in bovine livestock. The achievement of satisfactory results in TAI protocols depends of several factors, such as health and body condition, milk yield, environmental temperatures, ovulation time, semen manipulation, as well as semen quality. The goal of this study was to evaluate the conception rate of heifers and multiparous cows based on different Holstein bulls used for artificial insemination (AI). The effects of the type of management AI, animal category, and season of the year were also examined.Materials, Methods & Results: A three-year database on the reproductive management of a dairy farm was formed. All the cows and heifers were of the Holstein breed, in semi-intensive nutritional management, milked twice a day, and received a total mix ration. After 45 days in milk, when cows were observed twice daily with estrus signs, posterior artificial insemination occurred 12 h after estrus visualization was made. However, cows that were not observed in estrus were submitted to a timed artificial insemination (TAI). In this study, these fixed factors were considered: bulls utilized for the AI (totalizing 10 animals with at least 100 inseminations in each season, denoted by the letter B and a number - B1, B2, B3, B4, B5, B6, B7, B8, B9, B10); the season of the insemination (spring/summer being from November to April and autumn/winter from May to October), category animal (heifers, animals that never have been calved, or multiparous, animals that have already given birth more than once), and the type of AI (which included TAI using a protocol for synchronization, but cows having not necessarily shown estrus signs at the AI; ETAI: using protocol of synchronization, cows showing estrus signs at the AI; and AI with estrus visualization, without protocol of synchronization when animals were observed in estrus and were inseminated 12 h later. The fertility parameter of the bulls and reproductive performance in AI was based on conception rate. There was a statistical difference among different bulls in the same season (P ≤ 0.05). There was also a difference in reproductive performance of the same bulls between seasons, autumn/winter and spring/summer; B8 37.8% vs. 32.0% (P = 0.05) and B10 35.1% vs. 20.7% (P = 0.003). Regarding the individual animal category, only one bull (B6) demonstrated higher reproductive performance when utilized in the AI of heifers than multiparous 39.2% vs. 27.2% (P = 0.01). Similarly, regarding the effect of the type of AI, only one bull (B8) exhibited different results, obtaining better results for conception rate when utilized in fixed TAI with estrus visualization (35.5%) and in the AI with estrus visualization (39.0%) compared with that of TAI (19.9%) (P < 0.05).Discussion: From these results, it was observed that some bulls exhibited better performance in situations of greater challenge. In addition, some animals presented variation in efficiency with the demonstration of estrus or not. Therefore, bulls that present semen with higher fertility in certain types of AI could be utilized on a larger scale for to increase the reproductive rates in artificial insemination. The results of this study indicate that despite the tests indicating the semen that is submitted is adequate, there are still variations in quality and the reproductive efficiency of each bull. With the effects varying based on fertility and on different environmental and management situations.

Download Full-text

Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽

10.3390/molecules26144221 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4221

Author(s):

Aage Kristian Olsen Alstrup ◽

Svend Borup Jensen ◽

Ole Lerberg Nielsen ◽

Lars Jødal ◽

Pia Afzelius

Keyword(s):

Animal Model ◽

Animal Models ◽

Porcine Model ◽

Animal Experiments ◽

Radioactive Tracers ◽

The Individual ◽

Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

Download Full-text