Semen collection, characterisation and artificial insemination in the beluga (Delphinapterus leucas) using liquid-stored spermatozoa

2008 ◽  
Vol 20 (7) ◽  
pp. 770 ◽  
Author(s):  
J. K. O'Brien ◽  
K. J. Steinman ◽  
T. Schmitt ◽  
T. R. Robeck
Keyword(s):  
Artificial Insemination ◽  
Storage Temperature ◽  
Sperm Concentration ◽  
Serum Testosterone ◽  
Delphinapterus Leucas ◽  
Short Term ◽  
Preservation Method ◽  
First Time ◽  
Pregnancy And Birth

Ejaculates were collected from a beluga (Delphinapterus leucas) to gain an understanding of sperm biology and develop a short-term sperm preservation method for use in artificial insemination (AI). Ejaculate parameters and biochemistry, semen production and serum testosterone concentrations of an adult male were characterised for 21 months. Sperm viability, acrosome integrity and morphology did not change (P > 0.05) but ejaculate volume, sperm concentration and total spermatozoa per ejaculate were higher (P < 0.05) from January to June than from July to December. Peak testosterone concentrations (P < 0.05) were observed from October to April (8.0 ± 1.6 ng mL–1). The effects of hyaluronic acid (HA), antioxidants, storage temperature and time on in vitro sperm characteristics were examined. Motility parameters and viability were improved (P < 0.05) when semen was stored at 5°C compared with 21°C. During the first 24 h of storage sperm agglutination was absent only at 5°C in the presence of HA. A nulliparous 28-year-old female was inseminated endoscopically with liquid-stored semen. A pregnancy and birth of a calf was achieved following AI for the first time in this species, thereby validating both the AI technique and the fertility of beluga spermatozoa after chilled storage in a specialised diluent.

Preservation of beluga (Delphinapterus leucas) spermatozoa using a trehalose-based cryodiluent and directional freezing technology

2010 ◽  
Vol 22 (4) ◽  
pp. 653 ◽  
Author(s):  
J. K. O'Brien ◽  
T. R. Robeck
Keyword(s):  
Freezing Rate ◽  
Slow Freezing ◽  
Delphinapterus Leucas ◽  
Freezing Method ◽  
Progressive Motility ◽  
Sperm Characteristics ◽  
Preservation Method ◽  
Systematic Development ◽  
Directional Freezing

A beluga (Delphinapterus leucas) sperm preservation method was developed for use in genome banking and AI. In Study 1, glycerol-based cryodiluents (modified BF5F and modified Platz Diluent Variant (PDV)) were unable to maintain adequate progressive motility using straws (fast and slow freezing rate (FR)) or pellets (slow FR). Neither freezing method nor FR affected in vitro sperm characteristics (P > 0.05), but retention of prefreeze progressive motility following thawing was greater (P < 0.05) for BF5F (21%) than PDV (15%). In Study 2, examining the effects of straw freeze–thawing using BF5F with glycerol (1 and 3%, v/v) or trehalose (46 and 91 mM) on sperm characteristics, samples cryopreserved in trehalose exhibited superior (P < 0.05) in vitro parameters compared with their glycerol-treated counterparts. In Study 3, compared with a straw method, directional freezing using 91 mM trehalose enhanced (P < 0.05) sperm characteristics, with samples retaining 38%, 75% and 61% of their prefreeze progressive motility, curvilinear velocity and viability, respectively. A higher (P < 0.05) proportion of motile spermatozoa displayed rapid velocity after directional (21 ± 1%) compared with straw (12 ± 3%) freezing. Systematic development of a cryodiluent and the use of directional freezing resulted in beluga spermatozoa exhibiting adequate post-thaw quality for genome banking and use in AI.

158 FIRST LLAMA BORN BY IN VITRO FERTILIZATION

2017 ◽  
Vol 29 (1) ◽  
pp. 188 ◽  
Author(s):  
L. Landeo ◽  
J. Mendoza ◽  
L. Manrique ◽  
E. Taipe ◽  
R. Molina ◽  
...  
Keyword(s):  
Drug Release ◽  
Pregnancy Rate ◽  
Sperm Concentration ◽  
Cumulus Cells ◽  
South American ◽  
Vitro Fertilization ◽  
South American Camelids ◽  
Intravaginal Device ◽  
Pregnancy And Birth

The aim was to transfer alpaca and llama embryos obtained by IVF into recipient llamas and evaluate pregnancy and birth rates. Gametes were collected from samples of ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered by aspiration of ovarian follicles using a 5-mL syringe, where oocytes with at least 3 layers of cumulus cells and a homogenous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.1 IU), and oestradiol 17β for 30 and 36 h with 5% CO2 in air, for alpaca and llama, respectively. After the maturation time, COC were placed in FERT-TALP solution 30 min before in vitro insemination with epididymal sperm of alpaca and llama as appropriate, the sperm were selected by swim-up method with centrifuging twice in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-free BSA, in an incubator with 5% CO2 in air set at 39°C for 45 min, oocytes were co-incubated with sperm concentration of 3 × 106 mL−1 for 18 to 20 h at 39°C with 5% CO2 in air. The IVF was carried out the day of ovulation induction of recipients. A group of 15 morphologically intact Day-8 blastocysts derived from in vitro culture with SOF serum were transferred nonsurgically into the uterine horn ipsilateral to the corpus luteum of 15 synchronized recipient llamas with an intravaginal device (controlled internal drug release) for 8 days. Then, 6 days after controlled internal drug release removal, ovulation was induced in recipients with the application of 1 mL of GnRH with previous ultrasound confirmation of the presence of a dominant follicle greater than 7 mm in diameter. Nine alpaca blastocysts and 6 llama blastocysts were transferred. The pregnancy rate was assessed by ultrasound at 45 days after transfer. The results obtained were as follows: for pregnancy rate, 33.3% (3/9) and 50% (3/6) for alpaca and llama embryos, respectively; for birth rate, 0.0% (0/9) and 16.7% (1/6) for alpaca and llama embryos, respectively. An alpaca fetus and 2 llama fetuses were aborted between 7 and 10 months of pregnancy, and only a llama gestation ended successfully, producing the first birth of the world of a llama bred by IVF in South American camelids, demonstrating that it is possible to obtain viable offspring in these species using this biotechnology.

18 EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5°C AND 15°C

2011 ◽  
Vol 23 (1) ◽  
pp. 115 ◽  
Author(s):  
Z. Namula ◽  
R. Kodama ◽  
Y. Kaedei ◽  
F. Tanihara ◽  
V. L. Vien ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Storage Temperature ◽  
Semen Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Control Group ◽  
Boar Semen ◽  
Boar Spermatozoa

Liquid preservation of semen can be an alternative to frozen–thawed semen for artificial insemination. The success of a selection of boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the effects of skim milk on the viability and in vitro fertility of boar spermatozoa preserved in Modena-based extenders at 5°C and 15°C for 2 weeks. A total of 7 ejaculates were collected from one boar. The sperm-rich fraction of each ejaculate was centrifuged and diluted in Modena extenders supplemented with 0 (control), 7.5, and 15 mg mL–1 of dry skim milk. The final sperm concentration was adjusted to 1 × 108 cells mL–1, and then the semen was stored at 5°C and 15°C for 2 weeks. In the first experiment, the motility, viability (live/dead fluorescence viability assay), plasma membrane integrity (hypoosmotic swelling test; HOST), and acrosome integrity (FITC-labelled peanut agglutinin staining) of semen stored for 2 weeks were assessed. In the second experiment, the fertilization of stored semen after 20 h of co-incubation with in vitro matured oocytes and their development were examined. Data were analysed using ANOVA. When the semen was stored at 5°C for 2 weeks, the mean total sperm motility of semen stored with 7.5 and 15 mg mL–1 of dry skim milk was significantly higher than that of semen in the control group (41.4% and 41.5% v. 17.4%; P < 0.05). However, the beneficial effects of skim milk on the sperm motility were not observed in the semen stored at 15°C. Moreover, there were no significant differences in the other parameters of semen quality among the groups in each storage temperature. Significantly higher penetration rates of semen stored with 7.5 and 15 mg mL–1 of dry skim milk were observed in the storage at 5°C (41.1% and 34.8% v. 19.8%; P < 0.05) but not at 15°C (38.9% and 26.0% v. 30.0%; P > 0.05) when compared with the control group. When the semen was stored at 5°C, the development rate to the blastocyst stage of oocytes fertilized with semen stored with 7.5 mg mL–1 of dry skim milk was significantly higher than that with control and 15 mg mL–1 of dry skim milk (15.4% v. 1.1% and 7.8%; P < 0.01). However, there were no significant differences in the development rates of oocytes fertilized with semen stored at 15°C among the groups (9.6–11.9%). In conclusion, our results indicate that the effect of skim milk on the viability and in vitro fertility of liquid-stored boar spermatozoa is dependent on the storage temperature. The addition of 7.5 mg mL–1 of dry skim milk may be effective for the improvement of viability and fertility of semen stored at 5°C but not at 15°C.

Effects of short-term exposure to hydroxyflutamide in utero on the development of the reproductive tract in male mice

1995 ◽  
Vol 73 (11) ◽  
pp. 1582-1588 ◽  
Author(s):  
David W. Silversides ◽  
Christopher A. Price ◽  
Gerard M. Cooke
Keyword(s):  
Reproductive Tract ◽  
Serum Testosterone ◽  
Seminal Vesicles ◽  
Seminiferous Tubules ◽  
Long Term Effects ◽  
Short Term ◽  
Androgen Ablation ◽  
Androgen Action ◽  
Short Term Exposure

To determine if and when short-term ablation of androgen action compromises the development of the male reproductive tract in mice, the androgen receptor antagonist hydroxyflutamide was administered orally to pregnant FVB/N mice and the reproductive tracts of the male offspring were examined when adult. Hydroxyflutamide (30 mg per day) for 5 days from day 11 to day 15 of gestation caused hypospadias in all male progeny. However, testis weights, seminal vesicle weights, and serum testosterone levels were not affected (p > 0.05) but caput–corpus epididymal weights were 15% lower than controls (p < 0.02). Shorter periods of treatment that included day 14 or 15 caused hypospadias, but treatments that did not include days 14 and 15 did not (p < 0.002). Hydroxyflutamide (30 mg, once or twice daily for 2 consecutive days) between days 15 and 20 of gestation demonstrated that androgen ablation on days 15&16 caused hypospadias, absence of prostate, and scrotal location of the seminal vesicles with abdominal testes (p < 0.05). Males exposed later in pregnancy had prostates, but the weights were reduced (p < 0.001); testes were scrotal and seminal vesicles were abdominal; caput–corpus epididymal weights were 15–30% lower than controls (p < 0.05), but the tubule contained large numbers of spermatozoa. Furthermore, testis weights, serum testosterone, and the response of the testis to a human chorionic gonadotropin (hCG) challenge in vitro were not compromised by hydroxyflutamide, and seminiferous tubules exhibited normal spermatogenesis. When males that had been exposed to hydroxyflutamide on days 13&14, 15&16, 17&18, and 19&20 were housed with sexually mature females, pregnancies resulted only from the day 19&20 treatment group. Thus, there are long-term effects caused by short-term blockade of androgen action at critical times during pregnancy and such effects could result in the inability to impregnate, irrespective of any externally visible indications of developmental anomalies.Key words: antiandrogen, sexual development, fetus, male, mouse.

Slo3 K+ channel blocker clofilium extends bull and mouse sperm-fertilizing competence

Reproduction ◽  
2018 ◽  
Vol 156 (6) ◽  
pp. 463-476 ◽  
Author(s):  
Roland Abi Nahed ◽  
Guillaume Martinez ◽  
Jean Pascal Hograindleur ◽  
Emilie Le Blévec ◽  
Sabine Camugli ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Acrosome Reaction ◽  
Channel Blocker ◽  
Specific Inhibitor ◽  
K Channel ◽  
Sperm Dna ◽  
The Impact ◽  
First Time ◽  
Bovine Species

For artificial insemination (AI) to be successful, it is essential that sperm delivery be perfectly timed relative to ovulation, as sperm lifespan is limited due to oxidative metabolism induced by capacitation. Extending the window of sperm capacitation could therefore increase sperm lifespan, prolong sperm-fertilizing competence and increase AI efficiency. Hyperpolarization of sperm is a crucial step in capacitation and is induced by activation of the potassium calcium-activated channel subfamily U member 1 (KCNU1, also named Slo3 or KSper). Given the essential role played by KCNU1 in capacitation, this study assessed the impact of its pharmacological inhibition on sperm lifespan. We showed that treatment of murine sperm with sub-micromolar concentrations of clofilium, a specific inhibitor of KCNU1, slowed down capacitation, decreased the rate of acrosome reaction and extended the fertilizing competence of capacitated sperm for 12 h. Clofilium also extended fertilizing competence and motility of bovine-capacitated sperm, and increased the rate of fertilization with sperm capacitated for 24 h by 100%, and the rate of blastocyst formation by 150%. Finally, toxicity experiments showed clofilium to have no impact on sperm DNA and no embryotoxicity at the concentration used to extend sperm lifespan. Our results demonstrate that clofilium prolongs fertilizing competence of aging capacitated sperm in vitro in both rodent and bovine species. To our knowledge, this is the first time the duration of sperm-fertilizing competence is shown to be extended by potassium channels blockers.

scholarly journals Seminal Plasma, Sperm Concentration, and Sperm-PMN Interaction in the Donkey: An In Vitro Model to Study Endometrial Inflammation at Post-Insemination

2020 ◽  
Vol 21 (10) ◽  
pp. 3478 ◽  
Author(s):  
Jordi Miró ◽  
Henar Marín ◽  
Jaime Catalán ◽  
Marion Papas ◽  
Sabrina Gacem ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Artificial Insemination ◽  
In Vitro Model ◽  
Sperm Concentration ◽  
Polymorphonuclear Neutrophils ◽  
Low Fertility ◽  
Fertility Rates ◽  
Vitro Model ◽  
Instrumental Role

In the donkey, artificial insemination (AI) with frozen-thawed semen is associated with low fertility rates, which could be partially augmented through adding seminal plasma (SP) and increasing sperm concentration. On the other hand, post-AI endometrial inflammation in the jenny is significantly higher than in the mare. While previous studies analyzed this response through recovering Polymorphonuclear Neutrophils (PMN) from uterine washings, successive lavages can detrimentally impact the endometrium, leading to fertility issues. For this reason, the first set of experiments in this work intended to set an in vitro model through harvesting PMN from the peripheral blood of jennies. Thereafter, how PMN, which require a triggering agent like formyl-methionyl-leucyl-phenylalanine (FMLP) to be activated, are affected by donkey semen was interrogated. Finally, we tested how four concentrations of spermatozoa (100 × 106, 200 × 106, 500 × 106 and 1000 × 106 spermatozoa/mL) affected their interaction with PMN. We observed that semen, which consists of sperm and SP, is able to activate PMN. Whereas there was a reduced percentage of spermatozoa phagocytosed by PMN, most remained attached on the PMN surface or into a surrounding halo. Spermatozoa not attached to PMN were viable, and most of those bound to PMN were also viable and showed high tail beating. Finally, only sperm concentrations higher than 500 × 106 spermatozoa/mL showed free sperm cells after 3 h of incubation, and percentages of spermatozoa not attached to PMN were higher at 3 h than at 1 h, exhibiting high motility. We can thus conclude that semen activates PMN in the donkey, and that the percentage of spermatozoa phagocytosed by PMN is low. Furthermore, because percentages of spermatozoa not attached to PMN were higher after 3 h than after 1 h of incubation, we suggest that PMN-sperm interaction plays an instrumental role in the reproductive strategy of the donkey.

scholarly journals Reproductive parameters of Santa Inês ewes submitted to short-term treatment with re-used progesterone devices

2012 ◽  
Vol 64 (2) ◽  
pp. 333-340 ◽  
Author(s):  
A.E. Pinna ◽  
F.Z. Brandão ◽  
A.S. Cavalcanti ◽  
A.M. Borges ◽  
J.M.G. Souza ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Term Treatment ◽  
Reproductive Parameters ◽  
Device Removal ◽  
Short Term ◽  
Short Term Treatment ◽  
Response Interval ◽  
Santa Inês ◽  
First Time ◽  
Fresh Semen

The aim of this study was to verify the efficacy of reusing intravaginal progesterone (P4) devices on the reproductive parameters in Santa Inês ewes. Females received intravaginal P4 devices for their first, second or third use for five days plus 300 IU eCG IM and 5mg dinoprost laterovulvar 24h before device removal. Blood was collected at different moments. Transrectal ultrasonography was performed from device removal to ovulation. Part of the ewes were submitted to artificial insemination by laparoscopy (IAL - n=55) with fresh semen, whereas the rest were bred by fertile rams (n=41). On the initial 18 h, ewes that received devices for the first time showed higher P4 concentrations (5.1±1.8 vs 3.5±1.4 vs 2.4±1.1 - P<0.05). However, after the first 48h no difference was observed among all treatments and P4 supraluteal concentrations were detected in all ewes upon device removal. Estrous response, interval from device removal to estrus, rate of ovulating animals, number of ovulations, time from device removal to ovulation and average conception rates after IAL or natural mating were similar among all 3 groups. Intravaginal progesterone devices can be used up to three times without altering reproductive parameters in Santa Inês ewes.

Ultrastructural investigations of MDL 19,660-induced effects on the canine platelet and megakaryocyte

1990 ◽  
Vol 48 (3) ◽  
pp. 374-375
Author(s):  
D.E. Loudy ◽  
J. Sprinkle-Cavallo ◽  
J.T. Yarrington ◽  
F.Y. Thompson ◽  
J.P. Gibson
Keyword(s):  
Antidepressant Drug ◽  
Beagle Dogs ◽  
Long Term Effects ◽  
Short Term ◽  
Platelet Counts ◽  
Toxicological Studies ◽  
Induced Aggregation ◽  
Internal Architecture

Previous short term toxicological studies of one to two weeks duration have demonstrated that MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3Hl, 2,4-triazole-3-thione), an antidepressant drug, causes a dose-related thrombocytopenia in dogs. Platelet counts started to decline after two days of dosing with 30 mg/kg/day and continued to decrease to their lowest levels by 5-7 days. The loss in platelets was primarily of the small discoid subpopulation. In vitro studies have also indicated that MDL 19,660: does not spontaneously aggregate canine platelets and has moderate antiaggregating properties by inhibiting ADP-induced aggregation. The objectives of the present investigation of MDL 19,660 were to evaluate ultrastructurally long term effects on platelet internal architecture and changes in subpopulations of platelets and megakaryocytes.Nine male and nine female beagle dogs were divided equally into three groups and were administered orally 0, 15, or 30 mg/kg/day of MDL 19,660 for three months. Compared to a control platelet range of 353,000- 452,000/μl, a doserelated thrombocytopenia reached a maximum severity of an average of 135,000/μl for the 15 mg/kg/day dogs after two weeks and 81,000/μl for the 30 mg/kg/day dogs after one week.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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