Back-up frozen semen sample use in men planning on giving fresh semen samples for in vitro fertilization (IVF)

A 4-year-old beef bull underwent a breeding soundness evaluation at the Western College of Veterinary Medicine (Veterinary Hospital, University of Saskatchewan); no apparent abnormalities were observed after conventional semen evaluations. However, the clinical history of this bull indicated that no pregnancies resulted from natural service of 52 cycling cows over a period of 2 years. Completed services were observed. The objective of this study was to use in vitro fertilization (IVF) technology to evaluate whether the sperm from this infertile bull could successfully fertilize oocytes in vitro. Fresh semen was collected with an electroejaculator and frozen in a computer-controlled rate freezer. Concentration and motility parameters were assessed by using computer-assisted semen analyses. Sperm morphology was evaluated on eosin-nigrosin-stained slides, and Coomassie blue staining was used to evaluate the presence of intact acrosomes. Within each evaluation technique, frozen–thawed semen from bulls (n = 2) with proven fertility was used as a positive control and samples of dead sperm (produced by repeated frozen–thawed cycles until reaching no sperm motility) were used as a negative control. Frozen semen from the infertile bull was used for IVF assay. Data were analyzed by ANOVA (sperm motility and the presence of acrosomes) or the chi-square test (cleavage and blastocyst rates), with a P-value of 0.05. Our infertile bull showed an average motile sperm percentage of 91.7 ± 2.2%, with 78.6 ± 3.5% progressive motility. After cryopreservation procedures, frozen–thawed semen had an acceptable general and progressive percentage of motility of 57.8 ± 6.7% and 43.9 ± 9.2%, respectively. Sperm stained with Coomassie blue showed a greater proportion of intact acrosomes in the fresh semen (63.6 ± 4.3% v. 40.4 ± 3.7%; P < 0.05); however, frozen–thawed semen from both the fertile bull and the control were similar (40.4 ± 3.7b% v. 45.5 ± 2.2b%, P < 0.05). In vitro fertilization results revealed a low cleavage rate at 48 h postfertilization (19.8 ± 6.3%) and blastocyst rate (2.4 ± 2.8%) when using frozen–thawed semen from the infertile bull, compared with the control (56.7 ± 8.2% and 26.3 ± 4.5%, respectively; P < 0.001). Moreover, cleavage and blastocyst rates obtained by using the negative control (21.1 ± 3.2% and 1.1 ± 1.9%, respectively) were similar to those of the infertile bull (P > 0.10). It was noted that ova fertilized with either frozen–thawed semen from the infertile bull or the negative control did not produce blastocysts before Days 8 and 9 of embryo culture, which is a characteristic of parthenogenesis. The results from this IVF study suggest that in this bull, there was a missing or defective factor blocking one of the steps in the fertilization process. Further investigations to identify this factor will increase our knowledge of male fertility, and could lead to new methods of evaluating and regulating fertilizing ability.

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Comparison between the in vitro fertilization capability of frozen semen from different bulls

Theriogenology ◽

10.1016/s0093-691x(99)91887-3 ◽

1999 ◽

Vol 51 (1) ◽

pp. 328

Author(s):

G Rosés ◽

C Larocca ◽

I Lago ◽

J Calvo ◽

M Viqueira ◽

...

Keyword(s):

In Vitro Fertilization ◽

Vitro Fertilization ◽

Frozen Semen

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PSXI-10 How to use ultrasonic cutter on frozen semen for multiple embryo production

Journal of Animal Science ◽

10.1093/jas/skab235.454 ◽

2021 ◽

Vol 99 (Supplement_3) ◽

pp. 248-249

Author(s):

Sung Woo Kim ◽

Jae-Yeong Lee ◽

Chan-Lan Kim ◽

In-Sul Hwang ◽

Yeoung-Gyu Ko ◽

...

Keyword(s):

Survival Rate ◽

In Vitro Fertilization ◽

Endangered Species ◽

Genetic Resources ◽

Bovine Embryos ◽

Vitro Fertilization ◽

Economical Method ◽

Frozen Semen ◽

Ultrasonic Cutting

Abstract The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

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Is fresh or frozen semen to be used in in vitro fertilization with donor sperm?

Fertility and Sterility ◽

10.1016/s0015-0282(16)60617-9 ◽

1989 ◽

Vol 51 (4) ◽

pp. 661-664 ◽

Cited By ~ 23

Author(s):

Yvon. Englert ◽

Annick. Delvigne ◽

Marcel. Vekemans ◽

Bernard. Lejeune ◽

Alain. Henlisz ◽

...

Keyword(s):

In Vitro Fertilization ◽

Donor Sperm ◽

Vitro Fertilization ◽

Frozen Semen

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Comparative auto-controlled study between swim-up and Percoll preparation of fresh semen samples for in-vitro fertilization

Human Reproduction ◽

10.1093/oxfordjournals.humrep.a137657 ◽

1992 ◽

Vol 7 (3) ◽

pp. 399-402 ◽

Cited By ~ 47

Author(s):

Y. Englert ◽

M. Van den Bergh ◽

C. Rodesch ◽

E. Bertrand ◽

J. Biramane ◽

...

Keyword(s):

In Vitro Fertilization ◽

Controlled Study ◽

Vitro Fertilization ◽

Fresh Semen

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Role of back-up frozen semen in couples undergoing in vitro fertilization (IVF)

Fertility and Sterility ◽

10.1016/j.fertnstert.2004.07.708 ◽

2004 ◽

Vol 82 ◽

pp. S265

Author(s):

A.B. Pinto ◽

M.J. Putman ◽

S. Marynick ◽

N. Broyles ◽

R.D. Dickerson ◽

...

Keyword(s):

In Vitro Fertilization ◽

Vitro Fertilization ◽

Frozen Semen

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Spermatic feasibility after 20 years of freezing and refreezing: case study

Brazilian Archives of Biology and Technology ◽

10.1590/s1516-89132010000400014 ◽

2010 ◽

Vol 53 (4) ◽

pp. 851-854

Author(s):

Paulo Franco Taitson ◽

Maria do Carmo Borges de Souza

Keyword(s):

In Vitro Fertilization ◽

Testicular Cancer ◽

Vitro Fertilization ◽

Fresh Semen ◽

Sperm Freezing

The aim of the present work was to study the spermatic feasibility after 20 years of freezing, thawing and refreezing. A young man affected by testicular cancer sought the sperm freezing service. After 20 years, the samples were thawed and frozen again. After two days, thawing and analysis of the material took place. Comparison of the findings showed activity reduction from the first thawing to the fresh semen 59.09%. For the second thawing it was 77.27%. Considering the morphology, first thawing showed a reduction of 44.64% in relation to fresh semen. In the second thawing, the reduction was of 75%. Spermatic motility decreased by 64.28% in relation to fresh semen and 92.85% at the level of second thawing. The patient had gone through in vitro fertilization in 2007, obtaining success in the first one attempt.

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365 COMPARISON OF IN VITRO FERTILIZING CAPACITY OF FROZEN - THAWED SEX-SORTED AND SEX-SORTED FROZEN - THAWED BULL SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab365 ◽

2007 ◽

Vol 19 (1) ◽

pp. 298 ◽

Cited By ~ 3

Author(s):

V. Malcolm ◽

M. Marfil ◽

M. Calvi ◽

F. Rigali ◽

M. Pugliese ◽

...

Keyword(s):

In Vitro Fertilization ◽

Embryo Development ◽

Egg Yolk ◽

Reproductive Technologies ◽

Development Rate ◽

Vitro Fertilization ◽

Final Treatment ◽

Frozen Semen ◽

Sexed Semen

Sperm sexing has become a world-wide technology, now available in many countries. The method has been incorporated into many reproductive technologies such as embryo production (Zhang et al. 2003 Theriogenology 60, 1657–1663), but sex-sorting is limited when bulls are located far from sorters or when only frozen semen is available. Previous studies on sexing frozen–thawed spermatozoa have been done in rams, which resulted in retention of the spermatozoan functional capacities (Hollinshead et al. 2004 Reproduction 127, 557–568). In vitro characteristics were assessed in bulls after sexing of thawed sperm (Hollinshead et al. 2004 Theriogenology 62, 958–968); however, the fertilizing capacity of frozen–thawed sex-sorted (FTSS) spermatozoa was not tested. The aim of the present study was to compare cleavage and embryo development rate among frozen–thawed (FT), sex-sorted frozen–thawed (SSFT), and FTSS bull spermatozoa. For FT, sperm were diluted to a final concentration of 60 × 106 sperm/mL, packaged in 0.5-mL straws, and frozen. In SSFT, spermatozoa were sex-sorted by flow cytometry following Schenk protocols (1999 Theriogenology 52, 1375–1391). Three × 106 spermatozoa were packaged into 0.25-mL straws and frozen. The final treatment (FTSS) consisted of thawing 6 to 10 frozen straws of 4 different bulls containing an average of 25 × 106 spermatozoa and centrifuging at 600g for 15 min at 21°C to extract cryodiluent. Spermatozoa were diluted and stained with Hoechst 33342 (stain concentration of 112.5 µM, the same used for SSFT treatment) following Schenk sexed-semen protocols (1999), sex-sorted by a flow cytometer, and collected in Tris-base extender containing 20% egg yolk. For each ejaculate frozen–thawed, SSFT and FTSS spermatozoa were prepared for oocyte in vitro fertilization. Also, semen from a bull routinely used as a control in the laboratory was added for a better comparison of results. Oocytes from a slaughterhouse were processed following standard in vitro fertilization procedures (Ferré 2002 Theriogenology 57, 664) 4 times for each bull, and comparison was made between treatments. Results were analyzed by ANOVA. No significant differences were observed among bulls (data not shown) (P > 0.05). Although embryo development rate was statistically different between sexed and non-sexed groups (P < 0.05), results showed that frozen–thawed bull spermatozoa can be sex-sorted and used for in vitro fertilization with comparable developmental rates comparable to those when frozen sexed semen is used (Table 1). This opens a new commercial window for cases where pre-selected sexed embryos from bulls that are not in AI centers are desired, also giving an opportunity for dead bulls. Nevertheless, since a large number of straws are necessary, further studies must be carried out to make this procedure more efficient and economically profitable. Table 1. Results of cleavage and embryo development rates between spermatozoa treatments

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181 IN VITRO FERTILIZATION (IVF) USING SEMI-DEFINED CULTURE CONDITIONS FROM LOW OR HIGH ANTRAL FOLLICLE COUNT PUBERTAL BEEF HEIFERS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab181 ◽

2016 ◽

Vol 28 (2) ◽

pp. 221

Author(s):

C. C. Chase ◽

R. A. Cushman ◽

A. K. McNeel ◽

O. L. Amundson ◽

G. A. Perry ◽

...

Keyword(s):

In Vitro Fertilization ◽

Prostaglandin F2α ◽

Antral Follicle ◽

Antral Follicle Count ◽

Oestrous Cycle ◽

Vitro Fertilization ◽

Development Rates ◽

Frozen Semen ◽

Defined Culture

To compare the in vitro fertilization (IVF) and production (IVP) of embryos from low and high antral follicle count (AFC) heifers, AFC were determined on 106 heifers using transrectal ultrasonography. Ten heifers with the lowest AFC (avg. 13.2) and 10 heifers with the highest AFC (avg. 27.4) with evidence of oestrous cyclicity were synchronised with 2 injections of prostaglandin F2α (PGF2α); half were harvested on Days 5 to 6 and half on Days 15 to 16 of the oestrous cycle. The IVF procedures included protocols for semi-defined media and were as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida, Gainesville, FL, USA). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilised using thawed frozen semen from a crossbred bull that was purified using Percoll gradient separation procedures. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in microdrops of development medium under oil, and cultured (5% CO2, 5% O2, balance N2, 38.5°C). On Days 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the mixed procedure of SAS (SAS Institute, Cary, NC, USA) and the model included the effects of collection day, group (high or low AFC), and their interaction. More COC (P < 0.0005) were collected from high than low AFC heifers (30.3 v. 9.3 ± 3.12 per heifer). Both the number and percentage of COC that cleaved had an interaction between collection day and group (P < 0.03). The interaction appeared to be due to low cleavage and development rates on 1 of 4 collection days (appeared not related to day of oestrous cycle). Although high compared to low AFC heifers had more COC that cleaved (18.7 v. 4.4 ± 1.84 per heifer), the percentage of cleaved COC did not differ (59.2 v. 49.8 ± 3.36%). There were no significant differences between high and low AFC heifers in the number of blastocysts (3.1 v. 0.6 ± 1.21 per heifer) or in the percentage of COC that developed to blastocysts (8.8 v. 5.2 ± 3.60%). In previous replicates (years), we reported that high AFC heifers had more COC collected, more COC that cleaved, and more COC that developed to blastocysts than low AFC heifers. In contrast, in this study numbers of COC that developed to blastocysts did not significantly differ between high and low AFC heifers. Additionally, the percentage of COC that cleaved, and that developed to blastocyst have been similar between high and low AFC heifers. Therefore, high compared to low AFC heifers may produce more IVP embryos; however, AFC does not appear to affect the competence of an oocyte to develop and mature to the blastocyst stage.

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Ultrasonic Cutting of Frozen Semen Straws to Optimize the Use of Spermatozoa for In Vitro Fertilization

Animals ◽

10.3390/ani10112152 ◽

2020 ◽

Vol 10 (11) ◽

pp. 2152

Author(s):

Sung Woo Kim ◽

Jae-Yeong Lee ◽

Bongki Kim ◽

Chan-Lan Kim ◽

In-Sul Hwang ◽

...

Keyword(s):

Survival Rate ◽

In Vitro Fertilization ◽

Endangered Species ◽

Genetic Resources ◽

Bovine Embryos ◽

Vitro Fertilization ◽

Economical Method ◽

Frozen Semen ◽

Ultrasonic Cutting

The objective of the present study was to establish conditions for using technology that can potentially enhance the efficiency of bovine embryos derived from in vitro fertilization (IVF) with frozen semen. Frozen semen from selected bulls can be stored indefinitely in liquid nitrogen as genetic resources; however, these resources are considered consumable because they cannot be regenerated. Therefore, to optimize the utilization of frozen semen, as many oocytes as possible should be fertilized with one straw. However, a sufficient number of prepared oocytes might not be available for one experiment, which can limit the use of the total spermatozoa population. Thus, an economical method for producing embryos needs to be established by optimizing technology for transplantable embryos. In this study, the utilization of frozen semen was increased by dividing the straw with an ultrasonic cutter. The post-thaw survival rate of uncut straws from Korean Proven Bulls did not differ from that of half cuttings. When ultrasonic cutting was applied to frozen semen, spermatozoa could be prepared for IVF trials at least four times, and blastocysts were produced. Therefore, cutting frozen semen with an ultrasonic cutter represents a potentially useful tool to expand genetic resources from excellent breeding stocks. This approach could also be valuable in the field of IVF of endangered species or rare breeds for their preservation, as well as in ovum pick-up (OPU) techniques.

Download Full-text