Current status and advances in ram semen cryopreservation

Ram semen cryopreservation contributes to genetic improvement through artificial insemination, eliminates geographical barriers in artificial insemination application and supports the preservation of endangered breeds thus the conservation of biodiversity. Sperm freezing process induces ultrastructural, biochemical and functional changes of spermatozoa. Especially, spermatozoa’s membranes and chromatin can be damaged, sperm membranes’ permeability is increased, hyper oxidation and formation of reactive oxygen species takes place, affecting fertilizing ability and subsequent early embryonic development. Aiming to improve ram frozen-thawed semen’s fertilizing capacity, many scientific investigations took place. Among them the composition of semen extenders, was a main point of interest. Semen preservation extenders regulate and support an environment of adequate pH and buffering capacity to protect spermatozoa from osmotic and cryogenic stress. Therefore, permeating (glycerol, dimethyl sulfoxide) and non-permeat ing (egg yolk, skimmed milk) cryoprotectants, sugars (glucose, lactose, trehalose, raffinose), salts (sodium citrate, citric acid) and antioxidants (amino acids, vitamins, enzymes) have been added and tested. Moreover, semen dilution rate, storage temperature, cooling rate and thawing protocol, are also some key factors that have been studied. The research results of this scientific topic are encouraging, not only about the freezing and thawing procedures, but also about the improvement of the additives’ properties. However, further research is needed to enhance the fertilizing ability of ram frozen-thawed semen, making its use practical in sheep reproductive management by the application of cervical artificial insemination.

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Viability of Peranakan Etawah Liquid Semen Preserved in Tris Substituted with Various Energy Sources

Jurnal Ilmu Ternak dan Veteriner ◽

10.14334/jitv.v25i2.2026 ◽

2020 ◽

Vol 25 (2) ◽

pp. 68

Author(s):

Nurul Afzan Hilda Zakiya ◽

A H Yanti ◽

T R Setyawati

Keyword(s):

Energy Source ◽

Artificial Insemination ◽

Block Design ◽

Egg Yolk ◽

Energy Sources ◽

Randomized Block Design ◽

Frozen Semen ◽

Semen Preservation ◽

Semen Extender ◽

Fresh Semen

The use of liquid semen for artificial insemination program of Etawah crossbreed goat (PE) is an alternative to replace frozen semen which is constrained by limited and expensive facilities. Production of liquid semen is faster than frozen semen, but the viability of liquid semen which preserved with a standard extender such as tris egg yolk is very short. The purpose of this study was to determine the viability of PE goat semen in egg yolk tris substituted with energy sources such as glucose, galactose, and mannose and to determine the most efficient energy source for semen preservation. This research was conducted from August to September 2018 at the Artificial Insemination Center in Lembang, West Java. This study was designed in a randomized block design (RBD) consist of three experimental groups divided into five groups. Fresh semen of PE goats were preserved using extender which energy source has been modified. Results showed that using glucose in PE goat semen extender produced the best motility among other groups (64.29 ± 9.2%). The highest viability was found in extender with fructose substitution (86.76 ± 2.3%). The longest viability of liquid semen was found in the extender with glucose substitution. It lasted for six days.

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Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen

Semina Ciências Agrárias ◽

10.5433/1679-0359.2020v41n5supl1p2209 ◽

2020 ◽

pp. 2209-2218

Author(s):

Fernando Evaristo da Silva ◽

Jaqueline Candido Carvalho ◽

Camila de Paula Freitas Dell'Aqua ◽

Frederico Ozanam Papa ◽

Marc Roger Jean Marie Henry ◽

...

Keyword(s):

Cell Damage ◽

Membrane Integrity ◽

Sodium Caseinate ◽

Egg Yolk ◽

Freezing Process ◽

Computer Assisted ◽

Semen Cryopreservation ◽

Sperm Analysis ◽

Frozen Semen ◽

Buffalo Semen

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.

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Artificial insemination and cryopreservation of boar semen: current state and problematics

Open Science Journal ◽

10.23954/osj.v5i2.2353 ◽

2020 ◽

Vol 5 (2) ◽

Author(s):

Joaquim Moreira da Silva

Keyword(s):

Artificial Insemination ◽

Cold Shock ◽

Current Status ◽

Freezing And Thawing ◽

Swine Industry ◽

Current State ◽

Boar Semen ◽

The Many ◽

Cryopreservation Method

Commercial artificial insemination with boar semen still prefers the usage of refrigerated semen up to 5 days over frozen-thawed, to date. This is because of the uneconomical properties of frozen-thawed boar semen, such as low motility, viability, fertility rates and the need for higher semen doses, because of the decreased quality after cryopreservation. Since boar semen is highly susceptive to cold shock damage, the invention of a successful cryopreservation method would be greatly beneficial for the swine industry. This review briefly focuses on the many factors that influence the quality of frozen-thawed boar semen, including the different compositions of extenders, comparison of commercial extenders, freezing and thawing methods (temperature and duration). It could be concluded from the present review that optimum freezing/thawing protocol for swine is not standardized, so far being the current status still considered poor-to-fair.

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Determination of the optimal concentration of Minitube Equex paste for boar semen cryopreservation based on sperm motility characteristics

Veterinarska stanica ◽

10.46419/vs.52.3.8 ◽

2020 ◽

Vol 52 (3) ◽

Author(s):

Junpen Suwimonteerabutr ◽

Morakot Nuntapaitoon ◽

Padet Tummaruk

Keyword(s):

Sperm Motility ◽

Egg Yolk ◽

Group Iv ◽

Optimal Concentration ◽

Freezing And Thawing ◽

Semen Cryopreservation ◽

Group Iii ◽

Group I ◽

Boar Semen ◽

Group Ii

Equex paste is a non-permeating cryoprotective agent (CPA) that improved post-thaw survival of spermatozoa during boar semen cryopreservation. However, Equex paste produced by Nova Chemical Sales Inc. (MA, USA) is not currently available. The aim of the present study was to determine the optimal concentration of Minitube Equex paste (Minitube, Tiefenbach, Germany) for boar semen cryopreservation in comparison with Nova Equex STM paste (control). Fifteen ejaculates from 12 mature boars were collected by the glove-hand method. Each ejaculate was aliquoted and cryopreserved in base freezing extender III as Tris-citrate egg yolk (TEY) extender plus 9.0% glycerol classified into four groups. Group I was the control and included only 1.5% Nova Equex STM paste. Groups II, III and IV were the experiment groups, and contained different concentrations of Minitube Equex paste (Group II: 1.5%; Group III: 1.7%; and Group IV: 1.9%) added to the freezing extender III. After freezing and thawing, sperm motility characteristics were evaluated by Sperm Class Analyzer® incubated at 37 °C for 0 (10 min), 1 and 2 h post-thawing. In Group IV after thawing at 0 h, rapid velocity and the velocity curved line were significantly higher than in Groups II and III (P < 0.05) but did not differ from Group I. Moreover, after thawing at 1 h, LIN (linearity) in Group IV was higher than in Group II (P < 0.05), but did not differ from the other groups. In conclusion, the most suitable concentration of Minitube Equex paste in the current protocol was 1.9% supplemented with 9.0% glycerol in TEY-based freezing extender III, based on the conformity between data from manual guides and the observed sperm motility characteristics results.

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220 LIVE BIRTH OF A MOHOR GAZELLE (GAZELLA DAMA MHORR) CALF FOLLOWING INTRAUTERINE INSEMINATION OF MOTHER WITH FROZEN - THAWED SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab220 ◽

2006 ◽

Vol 18 (2) ◽

pp. 218 ◽

Cited By ~ 2

Author(s):

J. J. Garde ◽

M. Gomendio ◽

G. Espeso ◽

E. R. S. Roldan

Keyword(s):

Endangered Species ◽

Artificial Insemination ◽

Intrauterine Insemination ◽

Live Weight ◽

Egg Yolk ◽

Freezing And Thawing ◽

Technical Problems ◽

In The Wild ◽

Reproductive Techniques

Gazella dama mhorr is an endangered species, and no animals have been observed in the wild since 1968. Assisted reproductive techniques have been used to propagate endangered species. However, no live offspring has been produced after cryopreservation of semen from gazelle species. Therefore, the objective of this study was to evaluate whether cryopreserved Mohor gazelle spermatozoa can fertilize in vivo after artificial insemination. Semen was collected by electroejaculation from four males and centrifuged at 700g for 5 min at room temperature. The supernatant was discarded, and the semen pellet was resuspended with a TEST-1% egg yolk diluent containing 6% glycerol to provide 400 � 106 spermatozoa/mL. The extended semen was loaded into 0.25-mL plastic straws, cooled slowly to 5�C, and equilibrated at 5�C for 2 h. Straws were frozen in nitrogen vapors for 10 min and then plunged into liquid nitrogen. After thawing (37�C, 30 s), the effects of cryopreservation on sperm motility and acrosomal integrity were examined. Percentage of motile sperm in fresh samples was 88.7 � 3.8% (mean � SEM), decreased (P < 0.0001) to 58.7 � 3.8% after freezing and thawing, and then to 40.0 � 3.8% after 120 min incubation at 37�C. Spermatozoa with normal acrosomes decreased (P < 0.0001) after freezing and thawing (from 94.5 � 4.2% to 56.0 � 4.2%), but did not significantly decrease after sperm incubation. Frozen spermatozoa from two males were used in an intrauterine insemination trial. Estrus of females (n = 15) was synchronized with controlled internal drug release (CIDR, InterAg, Hamilton, New Zealand). Single CIDRs (type G, 330 mg progesterone/device) were inserted intravaginally for a total of 13 days. On Day 10, devices were replaced in each animal and all females received an injection of prostaglandin F2� (PGF2�; 125 mg/female). At CIDR withdrawal, females received 250-300 IU equine chorionic gonadotropin (eCG: Folligon; Intervet, Salamanca, Spain). After anesthesia with an intravenous injection of xylazine hydrochloride (Rompun; Bayer, Madrid, Spain; 0.8 mg/kg live weight) and ketamine hydrochloride (Imalgene; Leti & Merieux, Madrid, Spain; 2.0 mg/kg live weight), eight females were inseminated with 100 � 106 frozen-thawed spermatozoa 56-57 h after removal of the CIDRs, and seven were inseminated after 64-65 h. Females were inseminated directly into the uterus using laparoscopy. Anesthesia was reversed with yohimbine hydrochloride (0.3 mg/kg live weight). One female in the second group became pregnant. After a 202-day gestation, the female gave birth to one healthy Mohor gazelle male calf. These results demonstrate for the first time the successful use of frozen-thawed semen by means of artificial insemination for the rescue of endangered gazelle species. However, our results reveal that a number of unresolved technical problems remain to be addressed. This work was supported by the Spanish Ministry of Education and Science (REN2003-1587).

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Cryopreservation and fertility of frozen thawed Chegu goat semen

Indian Journal of Animal Research ◽

10.18805/ijar.b-3696 ◽

2019 ◽

Author(s):

A. Sharma ◽

P. Sood

Keyword(s):

Endangered Species ◽

Artificial Insemination ◽

Arid Region ◽

Egg Yolk ◽

Conception Rate ◽

Semen Parameters ◽

Semen Cryopreservation ◽

Livestock Species ◽

Progressive Motility ◽

Seminal Parameters

Goats are important livestock species of India. Chegu is a pashmina producing goat native to the cold arid region of Himachal Pradesh (H.P.), India. Semen cryopreservation from six Chegu elite males (aged 2.05±0.40 years; weighed 29.16±2.02 kg) was practiced using Tris Citrate Egg Yolk extender containing 10% EY and 6% Glycerol. Gross semen parameters includes volume (0.80.85±0.07 ml), color (Creamy white to yellowish), concentration (2238.5±231.0 x106 spermatozoa/ml) and mass motility (3.92±0.03).The significant changes (P less than 0.01) in post thaw seminal parameters (75.48±0.69 v/s 37.38±0.90; progressive motility), viability (75.79±0.95 v/s 48.25±1.78), morphological abnormalities (5.64±0.29 v/s 7.02±0.32) and HOST reactive spermatozoa (64.07±1.75 v/s 43.35±1.79) were observed in present study. Artificial insemination using frozen thawed semen having concentration (150 x106 spermatozoa/straw) from three different bucks was practiced in 40 synchronized goats with conception rate of 42.5 per cent. Non-significant variations amongst different bucks were observed with birth of 1.12 kids per doe and twinning rate of 11.8 per cent. It was concluded that semen cryopreservation along with artificial insemination can be practiced in Chegu goats to improve the population of this endangered species.

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Efficiency of Tris-Based Extender Steridyl for Semen Cryopreservation in Stallions

Animals ◽

10.3390/ani10101801 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1801

Author(s):

Elena Nikitkina ◽

Artem Musidray ◽

Anna Krutikova ◽

Polina Anipchenko ◽

Kirill Plemyashov ◽

...

Keyword(s):

Artificial Insemination ◽

Egg Yolk ◽

Dna Integrity ◽

Normal Morphology ◽

Progressive Motility ◽

Frozen Semen ◽

Fertilizing Ability ◽

Motility Of Sperm ◽

In Pregnancy ◽

Stimulation Of

The fertilizing ability of stallion sperm after freezing is lower than in other species. The search for the optimal extender, combination of extenders, and the freezing protocol is relevant. The aim of this study was to compare lactose-chelate-citrate-yolk (LCCY) extender, usually used in Russia, and Steridyl® (Minitube) for freezing sperm of stallions. Steridyl is a concentrated extender medium for freezing ruminant semen. It already contains sterilized egg yolk. Semen was collected from nine stallions, aged from 7 to 12 years old. The total and progressive motility of sperm frozen in Steridyl was significantly higher than in semen frozen in LCCY. The number of spermatozoa with normal morphology in samples frozen in LCCY was 60.4 ± 1.72%, and with Steridyl, 72.4 ± 2.10% (p < 0.01). Semen frozen in Steridyl showed good stimulation of respiration by 2.4-DNP, which indicates that oxidative phosphorylation was retained after freezing–thawing. No differences among the extenders were seen with the DNA integrity of spermatozoa. Six out of ten (60%) mares were pregnant after artificial insemination (AI) by LCCY frozen semen, and 9/12 (75%) by Steridyl frozen semen. No differences among extenders were seen in pregnancy rate. In conclusion, Steridyl was proven to be a good diluent for freezing stallion semen, even though it was developed for ruminants.

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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.

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Oestrous synchronization, semen preservation and artificial insemination in the Mohor gazelle (Gazella dama mhorr) for the establishment of a genome resource bank programme

Reproduction Fertility and Development ◽

10.1071/rd9961215 ◽

1996 ◽

Vol 8 (8) ◽

pp. 1215 ◽

Cited By ~ 50

Author(s):

WV Holt ◽

T Abaigar ◽

HN Jabbour

Keyword(s):

Endangered Species ◽

Artificial Insemination ◽

Egg Yolk ◽

Lauryl Sulfate ◽

Adult Males ◽

Technical Problems ◽

A Genome ◽

Semen Preservation ◽

Sperm Motion ◽

Extant Population

Gazella dama mhorr is an endangered species with an extant population of about 190 animals distributed between several zoos. Semen was collected by electro-ejaculation from 12 adult males, and cryopreserved in TEST-yolk diluent containing 6% glycerol. The effects of the concentration of egg yolk (5%, 10% and 20%) and the presence or absence of sodium triethanolamine lauryl sulfate (equex) on sperm motion and acrosomal integrity after thawing were examined. Increasing concentrations of egg yolk resulted in more acrosomal damage and poorer motility after thawing. The presence or absence of equex had no effect on either parameter. The frozen spermatozoa were used in an insemination trial, in which 13 females were treated with intravaginal progesterone-releasing devices to synchronize oestrus. Seven females were inseminated with frozen-thawed semen 48 h after removal of the devices, and six were inseminated after 60 h. Three females in the first group and one in the second group became pregnant. However, only one pregnancy (from the 48-h group) was carried to term. The study demonstrated the feasibility of applying artificial insemination in this species, but revealed that a number of outstanding technical problems remain to be solved.

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