Effect of Chicken Bone Extract Powder on Epididymal Sperm Quality of Male Wistar Rats

Calcium is one of the minerals that are essential for male reproductive function. Calcium deficiency adversely affects spermatogenesis, normal sperm function and results in infertility. The sperm quality of rats fed a standard diet containing chicken bone extract powder (BEP) was assessed in the present study. Twenty male 8-week-old rats, Wistar strain, were randomized by weight into two groups of ten rats each and fed ad libitum a standard diet containing calcium carbonate (CaCO3, control) or chicken BEP;  both were equivalent to 0.5% calcium. At the end of the 7-week consumption, the net body weight gains measured in control (101.33±21.81 g) and chicken BEP groups (100.74±26.80 g) were not significantly different (P0.05). The in vitro sperm quality in terms of concentration, motility, viability, resistance to hypotonic stress, acrosomal reaction ability and morphology was comparable between control and chicken BEP (all were P0.05). The results suggest that chicken BEP addition into feeds is an alternative calcium source that is as effective but less expensive as CaCO3, a commercial calcium (fortificant). At least, it has no detrimental effect on male reproductive function

Fertilization Rate and Number of Embryos on Day 2 after Intrauterine and Deep Intrauterine Insemination Using Frozen-Thawed Boar Semen in Multiparous Sows

The present study determines fertilization rate and number of embryos on Day 2 after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) using frozen-thawed (FT) boar semen in multiparous sows. Twelve crossbred Landrace × Yorkshire multiparous sows were included. The sows were inseminated at 24 h after oestrus detection and reinseminated every 12 h until ovulation took place. The inseminations were conducted using IUI with 2 × 109 FT sperm per dose (n = 6) and DIUI with 1 × 109 FT sperm per dose (n = 6). The sows were slaughtered at 45.1 ± 7.2 h after ovulation. Embryos and unfertilized oocytes were flushed from the oviducts. IUI yielded a better fertilization rate than DIUI (66.0% versus 31.0%, P < .001). The number of embryos was 13.5 ± 2.7 and 6.6 ± 3.2 embryos/sow in IUI and DIUI groups, respectively (P = .08). The proportion of sows having unilateral fertilization was higher in the DIUI (3/5) than the IUI group (1/6). In conclusion, IUI with at least 2 × 109 total number of FT boar spermatozoa is recommended

The effect of cryopreservation media on the quality of β-thalassemia mouse spermatozoa

Background: The mouse model of human diseases is commonly used for biomedical study, including β-thalassemia (β-thal), an inherited hemoglobin disorder. Maintaining the mice strain by natural mating systems is costly and seems impractical, especially during the COVID-19 pandemic. Sperm-freezing is a cost-effective solution for β-thal mouse colony management. Aim: To determine appropriate cryopreservation media for β-thal mouse spermatozoa to establish a β-thal mouse sperm bank. Methods: The epididymal spermatozoa of C57BL/6 wild-type (WT) and β-globin gene knockout thalassemia (BKO) mice were frozen in four freezing media: I) raffinose–skim milk–monothioglycerol (MTG), II) raffinose–skim milk– glutamine, III) raffinose–egg yolk–glycerol, and IV) egg yolk–TES–Tris. The sperm quality was assessed prior to and following freeze-thawing. Results: Compared with WT counterparts, the viable spermatozoa before freezing exhibiting elevated levels of oxidative stress were significantly greater in BKO (p = 0.01). After thawing, the membrane integrity of BKO spermatozoa preserved in I was significantly lower (p = 0.001). The sperm viability and membrane integrity of BKO males were also inferior when media III and IV were used (p = 0.008–0.027). The amount of oxidative stress in the spermatozoon of BKO mice was significantly greater when preserved in I, III, and IV (p = 0.002–0.044). Comparing freezing media, the motility and acrosome integrity of WT and BKO spermatozoa preserved in IV were significantly higher than those in other media (p &lt; 0.001 to p = 0.01). Spermatozoa with the highest mitochondrial membrane potential were observed in I in both genotypes (p = 0.012 to p &gt; 0.05). The viability, membrane integrity, and oxidative stress of post-thaw BKO spermatozoa did not significantly differ among freezing solutions. Conclusion: Irrespective of freezing media, spermatozoa of BKO males are rather more sensitive to cryopreservation than those of WT. Raffinose–skim milk–MTG/glutamine, raffinose–egg yolk–glycerol, and egg yolk–TES–Tris can all be used to preserve BKO mouse spermatozoa. However, with slightly better sperm characteristics, egg yolk–TES– Tris may be a diluent of choice for BKO mouse sperm cryopreservation. The addition of a reducing agent to thawing media is also strongly recommended to efficiently prevent oxidative stress and therefore improve frozen-thawed sperm survival

Komparativna studija dviju različitih metoda određivanja integriteta akrosome smrznute pa odmrznute sperme nerasta: bojanje s FITC-PNA/EthD-1 u usporedbi s bojanjem Coomassie plavom

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P>0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P>0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.Postoje izvješća da je bojanje Coomassie plavom učinkovita i jeftina metoda procjene integriteta akrosome spermija. Međutim, ne postoje izvješća o bojanju Coomassie plavom za procjenu integriteta akrosome krioprezervirane sperme nerasta. Nadalje, informacije u svezi podudarnosti između bojanja Coomassie plavom i fluorescein izotiocijanatom konjugiranim s aglutininom kikirikija i etidij homodimerom (FITC-PNA/ EthD-1) za procjenu integriteta akrosome spermija nisu dostupne niti za jednu vrstu. Stoga je ova studija provedena za određivanje učinkovitosti i podudarnosti između metoda bojanja Coomassie plavom i bojanja FITC-PNA/EthD-1 za procjenu integriteta akrosome smrznute pa odmrznute sperme nerasta. U eksperiment je uključeno ukupno 25 uzoraka sjemena. Sjeme je krioprezervirano uporabom razrjeđivača na bazi laktoze i žumanjka i pohranjeno u 0,5 PVC-pajete. Svojstva pokretljivosti i gibanja spermija ustvrđena su uporabom sustava za računalno potpomognutu analizu spermija. Vijabilnost spermija i integritet njihove stanične membrane procijenjeni su uporabom SYBR-14/EthD-1, odnosno hipoosmotskim testom bubrenja. Integritet akrosome smrtznute pa odmrznute sperme nerasta procijenjen je uporabom bojanja FITC-PNA/EthD-1 i Coomassie plavom. Procijenjena je povezanost između integriteta akrosome sperme ustvrđene pomoću bojanja FITC-PNA/EthD-1 i Coomassie plavom te podudarnost između dviju metoda mjerenja. Prosječni postotak integriteta akrosome smrznute pa odmrznute sperme nerasta ustvrđene bojanjem FITC-PNA/EthD-1 i Coomassie plavom bio je 48,8 ± 12,6 %, odnosno 52,6 ± 13,6% (P>0,05). Zanimljivo, vijabilnost spermija korelirala je s integritetom akrosome procijenjenom uporabom Coomassie plavom (r=0,609, P=0,002), ali nije korelirala kada je procjenjivana uporabom FITC-PNA/EthD-1 bojanja (P>0,05). Međutim, integritet akrosome smrznute pa odmrznute sperme nerasta procijenjen bojanjem FITC-PNA/EthD-1 i Coomassie plavom značajno je korelirao (r=0,448, P=0,025, n=25). Uz to, podudarnost između bojanja Coomassie plavom i FITC-PNA za procjenu integriteta akrosome smrznute pa odmrznute sperme nerasta ustvrđena Bland- Altmanovim grafikonom bila je prihvatljiva. Zaključno, integritet akrosome smrznute pa odmrznute sperme nerasta procijenjena bojanjem Coomassie plavom značajno je korelirao s onim dobivenim FITC-PNA/EthD- 1 metodom bojanja te su te dvije metode pokazale dobru podudarnost. Uz to, značajna povezanost između integriteta akrosoma smrznute pa odmrznute sperme nerasta koja je ustvrđena bojanjem Coomassie plavom i drugih parametara kakvoće sperme ukazuju na to da je bojanje Coomassie plavom učinkovita metoda procjene integriteta akrosome smrznute pa odmrznute sperme u svinja

Razlika između proteina sjemene plazme i proteina sperme za dobru i lošu sposobnost smrzavanja ejakulata nerasta

The present study was performed to compare the expression of sperm proteins, i.e. triosephosphate isomerase (TPI) and acrosin binding protein (ACRBP) and seminal plasma proteins, i.e. glutathione peroxidase 5 (GPX5) and fibronectin 1 (FN1), in boar semen with good, moderate and poor freezability. The study was conducted by determining the protein contents in 32 sperm samples and 38 seminal plasma samples of semen. The ejaculated semen was divided into two portions: the first portion was centrifuged to separate the pellet of sperm from the seminal plasma and the second portion was cryopreserved. After thawing, the ejaculates were classified into three groups according to their post-thawed sperm motility: good (60.2 ± 1.7%), moderate (29.3 ± 2.0%) and poor (16.6 ± 2.2%) freezabilities. The expressions of GPX5 and FN1 in seminal plasma and TPI and ACRBP in sperm were determined using Western blot analysis. It was found that, for sperm proteins, the level of TPI was negatively correlated with the post-thawed total sperm motility (r = -0.38, P = 0.029). For seminal plasma proteins, the level of FN1 in the seminal plasma was positively correlated with the post-thawed total sperm motility (r = 0.37, P = 0.021) and progressive motility (r = 0.39, P = 0.016). The expression of GPX5 was not correlated with any of the frozen–thawed sperm qualities (P > 0.05). In conclusions, boar semen containing a high level of FN1 in seminal plasma has better freezability. Frozen–thawed sperm motility was positively correlated with the level of FN1 in boar seminal plasma and negatively correlated with TPI in boar spermatozoa.Ova studija provedena je u svrhu usporedbe ekspresije proteina sperme, tj. trioza-fosfat izomeraze (TPI) i akrozin-vezujućeg proteina (ACRBP) te proteina sjemene plazme, tj. glutation peroksidaze 5 (GPX5) i fibronektina 1 (FN1), u sjemenu nerasta s dobrom, umjerenom i lošom sposobnošću smrzavanja. Studija je provedena ustvrđivanjem sadržaja proteina u 32 uzorka sperme i 38 uzoraka sjemene plazme sjemena. Ejakulirano sjeme podijeljeno je u dva dijela: prvi dio je centrifugiran za odvajanje taloga sperme od sjemene plazme, a drugi je dio je krioprezerviran. Nakon odmrzavanja u skladu s pokretljivošću spermija nakon odmrzavanja ejakulati su klasificirani u tri skupine: dobra (60,2 ± 1,7%), umjerena (29,3 ± 2,0%) i loša (16,6 ± 2,2%) sposobnost smrzavanja. Ekspresije GPX5 i FN1 u sjemenoj plazmi te TPI i ACRBP u spermi ustvrđene su „Western blot“ analizom. Za proteine sperme je otkriveno da je razina TPI nakon odmrzavanja negativno povezana s ukupnom pokretljivošću sperme (r = -0,38, P = 0,029). Za proteine sjemene plazme, razina FN1 u sjemenoj plazmi nakon odmrzavanja pozitivno je povezana s ukupnom pokretljivošću sperme nakon odmrzavanja (r = 0,37, P = 0,021) i progresivnom pokretljivošću (r = 0,39, P = 0,016). Ekspresija GPX5 nije povezana ni sa kakvim kvalitetama smrznute pa odmrznute sperme (P > 0,05). Zaključno, sjeme nerasta koje sadrži visoku razinu FN1 u sjemenoj plazmi ima bolju sposobnost smrzavanja. Pokretljivost sperme koja je smrznuta pa odmrznuta pozitivno je povezana s razinom FN1 u sjemenoj plazmi nerasta, a negativno s razinom TPI u spermijima nerasta

The MTT assay application to measure the viability of spermatozoa: a variety of the assay protocols

The MTT reduction assay is one of the methods used to evaluate the viability of sperm. In the assay, a tetrazolium component (MTT) is converted into MTT formazan by some specific enzymes in the viable cells. The amount of formazan product in theory is directly correlated with the percentage of viable sperms. It is quantified by measuring the absorbance using a spectrophotometer. The present article compiles the MTT assays that have been used to determine sperm viability in most animal species and humans. In each assay, other factors apart from the number of viable cells that potentially influence the accuracy and precision of results are stated, such as preparations of sperm and MTT solutions, length and conditions of incubation, and a solubilizing agent as well as the formazan detection method. Also, the strengths and shortcomings of the MTT test comparison with the others are summarized at the end of this article. This information may be useful for prospective researchers deciding to implement this colorimetric method in their experiments

Male reproductive phenotypes of genetically altered laboratory mice (Mus musculus): a review based on pertinent literature from the last three decades

Laboratory mice (Mus musculus) are preferred animals for biomedical research due to the close relationship with humans in several aspects. Therefore, mice with diverse genetic traits have been generated to mimic human characteristics of interest. Some genetically altered mouse strains, on purpose or by accident, have reproductive phenotypes and/or fertility deviating from wild-type mice. The distinct reproductive phenotypes of genetically altered male mice mentioned in this paper are grouped based on reproductive organs, beginning with the brain (i.e., the hypothalamus and anterior pituitary) that regulates sexual maturity and development, the testis where male gametes and sex steroid hormones are produced, the epididymis, the accessory sex glands, and the penis which involve in sperm maturation, storage, and ejaculation. Also, distinct characteristics of mature sperm from genetically altered mice are described here. This repository will hopefully be a valuable resource for both humans, in terms of future biomedical research, and mice, in the aspect of the establishment of optimal sperm preservation protocols for individual mouse strains

Intra-uterine and Deep intra-uterine Insemination using Cryopreserved Boar Semen in Spontaneously-ovulating Sows บทคั ดย่ อ การผสมเที ยมแบบสอดท่ อเข้ าตั วมดลู กและปี กมดลู กด้ วยน้ ํ าเชื ้ อพ่ อสุ กรแช่ แข็ งในแม่ สุ กรที ่ ตกไข่ ตามธรรมชาติ คคนางค์ บู

Abstract The present study was performed to investigate the in vivo fertility of frozen-thawed (FT) boar semen after intra-uterine (IUI) and deep intra-uterine insemination (DIUI) in spontaneously-ovulating sows. A total of 48 weaned sows were included. The sows were divided into three groups, i.e. natural mating (NM) (n=30), IUI (n=9) and DIUI (n=9). In the IUI and DIUI groups, the sows were inseminated twice, at 24 and 36 h after the detection of oestrous by IUI with 2x10 9 spermatozoa/dose or DIUI with 1x10 9 spermatozoa/dose. Transrectal ultrasonography was used to determine the time of ovulation after insemination. The results revealed that the conception as determined by a 24-day non-return rate of the sows was 96.6%, 88.8% and 66.6% (p=0.03) and the farrowing rate (FR) was 96.6%, 66.6% and 66.6% (p=0.01) in NM, IUI and DIUI groups, respectively. The numbers of total piglets born per litter were 9.4±2.8, 11.3±2.9 and 7.6±3.1 piglets in the NM, IUI and DIUI groups, respectively (p=0.10). These data indicate that the spontaneously-ovulating weaned sows inseminated with either IUI or DIUI using a relatively low numbers of FT spermatozoa resulted in a lower FR compared to NM. The total number of piglets born per litter after IUI was higher than DIUI. Keywords: artificial insemination, frozen-thawed spermatozoa, ovulation, so