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While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE., Competing Interests: Declaration of competing interest The authors affirm that they have no competing or conflicts of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 °C to the freezing lab. On arrival, samples were split into the treatments with the following additions before cryopreservation; 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Semen was evaluated for motility kinetics at 30, 120, and 240 min after thawing. Flow cytometry assessments were performed at 60 min after thawing. At all-time points evaluated, total and progressive motility were greater (P ≤ 0.05) in semen cryopreserved with GSH than in Control. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity; however, the proportion of capacitated spermatozoa were reduced (by -21.17%) in semen treated with BHT + GSH compared to Control (P ≤ 0.05). In contrast, there was a higher (P ≤ 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. For IVF, semen cryopreserved with both antioxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (-54.11%) compared to Control. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P ≤ 0.05) for BHT compared to Control. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Progesterone (P4) plays a key role in pregnancy establishment and maintenance; during early pregnancy, P4 stimulates the production and release of uterine secretions necessary for conceptus growth prior to implantation; therefore, exogenous P4 supplementation may improve embryo development. This study evaluated the effects of supplementation during early pregnancy with long-acting injectable progesterone or altrenogest on embryonic characteristics of sows and gilts. Thus, a total of 32 sows and 16 gilts were used. On day 6 of pregnancy sows and gilts were allocated to one of the following groups: non-supplemented; supplemented with 20 mg of altrenogest, orally, from days 6 to 12 of pregnancy; supplemented with 2.15 mg/kg of long-acting injectable progesterone on day 6 of pregnancy. Animals were killed on day 28 of pregnancy, and ovulation rate, embryo survival, embryo weight, crown-to-rump length, uterine glandular epithelium and endometrial vascularization were assessed. Treatments had no effect on pregnancy rate, embryo survival or endometrial vascular density (P > 0.05). Non-supplemented gilts presented larger and heavier embryos compared to gilts from supplemented groups (P < 0.05). Sows in the altrenogest group presented larger and heavier embryos compared to non-supplemented sows and sows supplemented with long-acting injectable progesterone. In conclusion, supplementation of sows and gilts with progestagen from day 6 of pregnancy can be used as a means to improve embryo survival without deleterious effects.

Seminal plasma (SP) plays a vital role in the maintenance of sperm function and integrity along with being involved in their communication with the female reproductive tract. Under in vitro conditions, although it is generally accepted that boar semen is better preserved when SP is diluted (extended) or removed (cryopreserved), its role during storage is not completely elucidated. In this context, the current study sought to determine the role of SP during storage of boar spermatozoa at 17 °C for 72 h. Thus, two treatments were prepared with semen from the sperm-rich fraction (SRF) of boar ejaculate previous to storage in liquid state: 1) PSP: semen directly extended in Beltsville Thawing Solution (BTS), and 2) ASP: semen first centrifuged with subsequent removal of supernatant (containing SP) followed by suspension of sperm in BTS. From this, two experiments were conducted separately in this work: 1) in vitro and 2) in vivo assays. The former aimed to evaluate how sperm capacity responds to in vitro capacitation (IVC) and whether their quality is affected by previous exposure to SP. In the latter, the objective was to understand how important these previous conditions can be for reproductive performance after artificial insemination (AI). According to our results, the previous removal of SP does not affect sperm quality and the response of these cells to IVC (P > 0.05) along with resulting in a lower percentage of acrosome damage in them [12.87 ± 0.76 (ASP) vs. 16.38 ± 0.73 (PSP)] (P < 0.05). This improved preservation of acrosome integrity in the absence of SP can explain the higher fertility rate (%) [63.27 ± 23.47 (ASP) vs. 38.57 ± 16.30 (PSP)] and number of implanted embryos at 28 days after AI (13.71 ± 4.88 (ASP) vs. 7.16 ± 4.02 (PSP)] (P < 0.05) presented by gilts inseminated with seminal doses of ASP. In conclusion, removal of SP prior to liquid storage of boar sperm for 72 h can be beneficial for their fertilizing ability., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Boar semen cannot be immediately cryopreserved, it need be hold at 17 °C prior to cryopreservation, holding time has been used to improve cryopreserved boar semen, since holding time allows a prolonged interaction between spermatozoa and seminal plasma components. However, until now only few periods of holding time have been studied, and boar semen had been held at 17 °C for 24 h to facilitate its manufacture. Thus, this experiment aims to study the effect several holding time (0, 4, 8, 12, 24, 28 and 32 h) on boar spermatozoa post-thawed (PT) characteristics. Fifteen sperm-rich fractions of ejaculate were extended in Beltsville Thawing Solution and storage at 17 °C. After each holding time (0, 4, 8, 12, 24, 28 and 32 h), a sample was centrifuged, and sperm pellet was diluted in an extender composed of sugars, amino acids, buffers, 20% egg yolk (v/v), antibiotics, 2% glycerol as a cryoprotectant, and 2% methylformamide (v/v). Cryopreservation was performed with an automatic cryopreservation system. Cryopreserved boar semen was evaluated to spermatozoa kinetics, plasma and acrosomal membranes integrity, mitochondrial membrane potential, detection of superoxide anion, plasma membrane fluidity, and peroxidation. Twenty-four hours of holding increase total and progressive motility, rapid spermatozoa, and integrity of plasma and acrosome membranes. To mitochondrial membrane potential, 32 h is needed. However, holding time was not able to control the superoxide anion amount neither membrane lipid peroxidation, and had no effects on membrane fluidity. Thus, to reach the best results of PT boar semen the ideal holding time is 24 h., (Copyright © 2018 Elsevier Inc. All rights reserved.)