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

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).

2011 ◽  
Vol 23 (1) ◽  
pp. 146
Author(s):  
T. Okazaki ◽  
T. Akiyoshi ◽  
M. Kan ◽  
H. Teshima ◽  
M. Shimada

Epididymal spermatozoa are one of the available male germ cells for cryopreservation. It has been reported that frozen–thawed porcine epididymal spermatozoa have a high fertilization competence in vitro as compared with that in ejaculated one. However, there is little information about reproductive performance, such as conception rate or litter size, after artificial insemination (AI) using frozen–thawed epididymal spermatozoa. Recently, we demonstrated that the addition of seminal plasma to thawing solution improves membrane and acrosomal integrity, and enhanced both in vivo and in vitro fertilizing activity of frozen–thawed ejaculated spermatozoa. Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the number of implantation site (Okazaki et al. 2009 Theriogenology 71, 491–498). Thus, to apply those positive functions of seminal plasma to AI using frozen–thawed epididymal sperm, in this study, we added seminal plasma to thawing solution and then analysed the sperm functions including AI test using frozen–thawed epididymal spermatozoa. Epididymal spermatozoa collected by flushing caudal epididymis were frozen as described in our previous study (Okazaki et al. 2009). Frozen-spermatozoa were thawed in Modena solution with or without different percentages of seminal plasma. Protein tyrosine phosphorylation as a marker of capacitation was detected by western blotting. To examine the reproductive performance, the sows of natural oestrus were artificially inseminated two times (5 × 109 50 mL–1 per injection). When the frozen–thawed ejaculated or epididymal sperm was incubated up to 6 h, the motility of epididymal sperm was significantly higher than that of ejaculated sperm (19.6 v. 37.6%). However, the acrosomal membrane was damaged in epididymal sperm group at 3-h incubation period (15.2 v. 36.0%). The addition of seminal plasma [0, 10, 15, 20% (v/v)] in Modena solution protected the acrosomal injury (3 h; 35.2, 19.5, 15.6, 14.6%) and maintained high rate of motility (6 h; 38.8, 48.8, 62.5, 60.0%) in a dose-dependent manner. Furthermore, the addition of seminal plasma suppressed the expression of the 15 kDa phosphoprotein (early capacitation status), and the maximum effect was detected at 15% (v/v) seminal plasma. When the frozen–thawed epididymal spermatozoa with 15% (v/v) seminal plasma were artificially inseminated to swine (n = 15), the conception rate and the mean number of litter size were increased as compared with control (93 v. 43%, 10.0 v. 5.0). From these results, we concluded that the addition of seminal plasma to thawing solution was a beneficial method for artificial insemination using frozen–thawed epididymal spermatozoa in the pig. 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).


2021 ◽  
Vol 22 (9) ◽  
pp. 4368
Author(s):  
Heriberto Rodriguez-Martinez ◽  
Emilio A. Martinez ◽  
Juan J. Calvete ◽  
Fernando J. Peña Vega ◽  
Jordi Roca

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.


2021 ◽  
Author(s):  
Weihai Xu ◽  
Lin Zhang ◽  
Ling Zhang ◽  
Shishi Li ◽  
Jing Shu

Abstract Background: In this study, we compared the in vitro embryo development, embryo transfer outcome and the offspring outcome in the in vitro fertilization-embryo transfer (IVF-ET) between dry culture (DC) and humid culture (HC). Methods: Our study was divided into two parts. Firstly, we determined the fertilization rate, cleavage rate and high-quality embryo rate from 21 cycles in the DC group (N=262 oocytes) and HC group (N=263 oocytes). Secondly, we determined the embryo transfer outcome and the offspring outcome in DC group (N=184 cycles) and HC group (N=136 cycles). Results: Compared with the HC group, significant increase was observed in the high-quality embryo rate (66.1.2% vs. 55.3%, p=0.037) and implantation rate (49.8% vs. 40.6%, p=0.027) in the DC group. No statistical differences were observed in the pregnant outcome and birth defect of the offspring (p>0.05). Compared with HC, DC was associated with a higher high-quality embryo rate and a higher implantation rate after embryo transfer. Conclusions: No statistical differences were noticed in the offspring conditions between the two culture modes. Taken together, DC may serve as a promising method for IVF-ET.


2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
A. M. Taiyeb Ridha ◽  
D. C. Kraemer

In vitro synchronization of oocyte nuclear and cytoplasmic maturation has been found to improve the IVF rate of ovarian oocytes in several species, including humans, in comparison with nonsynchronized in vitro-matured oocytes. Here, we tested the hypothesis that synchronization of oocyte meiotic maturation by an in vivo system in superovulated mice would increase the oocyte fertilization rate when compared to that of conventional superovulated oocytes. Recently, we observed that cilostazol (CZL), a PDE3-I, was able to inhibit mouse oocyte meiotic maturation in both in vitro and in vivo systems. Administering CZL at 7.5 mg, 4 or 7 h pre-hCG allowed retrieval of ovulated oocytes of which >95% were at MI stage, scored by Nikon stereo microscope (SMZ 1500). A conventional superovulation program was adapted in all treated and their control groups, in which mice were injected with eCG and after 48 h with hCG (7.5 IU for each hormone). On the second morning, 13 to 14 h post-hCG, mice were killed and oocytes were collected from oviducts and in vitro fertilized (control). For the treated groups, CZL was administered in a single 7.5 mg oral dose (gavage) 4 or 7 h before the hCG injection. On the second morning, CZL-treated animals were killed at the same timing as control animals and oocytes were retrieved from the oviduct and in vitro matured for 6 h (for those gavaged with CZL, 4 h pre-hCG) or 3 h (for those gavaged with CZL, 7 h pre-hCG) to MII oocytes before IVF. These groups were designated as in vivo-in vitro synchronized/matured oocytes. In other groups treated with CZL, 4 or 7 h pre-hCG, the ovulated oocytes were allowed to mature in the oviduct (full in vivo synchronization and maturation) and oocytes were retrieved and fertilized with the same fertilization timings as the in vivo-in vitro synchronized/matured oocytes. Oocytes were cultured for 1 day after IVF and examined for cleavage. Statistical differences were analyzed by cross-tabulated chi-square test. The full in vivo synchronization and maturation (for both CZL dose timings of 4 and 7 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [89% (n = 219) and 92.2% (n = 374) vs 81.8% (n = 198); P = 0.034 and P < 0.0001, respectively]. The in vivo-in vitro synchronized/matured oocytes (CZL dose timing at 7 h, but not 4 h pre-hCG) gave significantly higher early embryonic development rates compared with those of the control [88.5% (n = 339) vs 83.4% (n = 458), respectively; P = 0.043]. However, the increase of the IVF rate of the oocytes from mice treated with CZL, 4 h pre-hCG, in the in vivo-in vitro synchronized/matured group was not significantly different from the control group [88.5% (n = 399) vs 83.4% (n = 458), respectively; P = 0.43]. It is concluded from the present study that synchronization of oocyte meiotic maturation by the in vivo and in vivo-in-vitro protocols can increase the IVF rate of oocytes in superovulated mice.


Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Maria Cristina Budani ◽  
Erminia Carletti ◽  
Gian Mario Tiboni

SummaryThis study was undertaken to evaluate whether cigarette smoke is associated with changes in the expression of antioxidant enzymes in granulosa cells of women undergoing IVF treatments. For this aim, the expression of three antioxidant enzymes (SOD1, SOD2 and catalase) in non-smokers (n = 20) and smokers (n = 20) was analyzed. There was a statistically significant overexpression of SOD2 and catalase mRNA levels in smokers in comparison with non-smokers. Cigarette smoking was associated with a lower fertilization rate, implantation rate and pregnancy rate in comparison with non-smokers. There was no effect on retrieved oocytes number, metaphase II oocytes number, quality of embryos transferred and live birth rate. These findings suggest that cigarette smoke initiates oxidative stress in granulosa cells.


Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.


2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P &lt; 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P &lt; 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.


Author(s):  
Iman Al-Saleh ◽  
Serdar Coskun ◽  
Reem Al-Rouqi ◽  
Tahreer Al-Rajudi ◽  
Chafica Eltabache ◽  
...  

This study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate, biochemical pregnancy, clinical pregnancy, and live birth). We observed an increase in the oxidative stress markers MDA, 8-OHdG, and H2O2 in follicular fluid and seminal plasma, but a decrease in the antioxidant protection markers CAT and TAC. The MDA, 8-OHdG, and H2O2 levels were significantly higher in seminal plasma than in follicular fluid, while TAC, CAT, and TM were higher in follicular fluid (p < 0.001). Although women were nonsmokers, COT levels >50 µg/l were observed in 5.7% (urine) and 1.4% (follicular fluid). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%).


2021 ◽  
Vol 26 (1) ◽  
pp. 7-14
Author(s):  
Mihai Cenariu ◽  
Mihai Borzan ◽  
Sorin Dan ◽  
Remus Chiorean ◽  
Emoke Pall

Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.


2013 ◽  
Vol 94 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Bao-Guo Xie ◽  
Yuan-Hua Huang ◽  
Wei-Jie Zhu ◽  
Song Jin

Objective: To evaluate whether couples with moderate male infertility should be treated with conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Patients and Methods: A total of 249 couples with moderate male infertility undergoing their first IVF/ICSI cycle were enrolled in the study. The couples were divided into two groups according to the results of semen analysis: moderate oligozoospermia (O group) and moderate oligoasthenozoospermia (OA group). Sibling oocytes were randomized into groups to be inseminated either by conventional IVF or ICSI. Fertilization rate, embryo quality, implantation rate, and clinical pregnancy rate were examined. Results: There was no difference in the fertilization, implantation, and pregnancy rates between conventional IVF and ICSI in either the O group or OA group (p > 0.05). Additionally, in the OA group, the good quality embryo rate was similar after IVF or ICSI (p > 0.05). However, in the O group, the good quality embryo rate was significantly higher after ICSI than after IVF (p < 0.05). Conclusions: Couples with moderate oligozoospermia or moderate oligoasthenozoospermia did not influence the major indices of IVF. Because of the uncertainties concerning the safety of ICSI, couples with moderate oligozoospermia or moderate oligoasthenozoospermia need not be subjected to this procedure.


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