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In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The addition of antioxidants to the cryopreservation medium has been shown to exert a positive effect on the quality of frozen-thawed sperm in different species. The objective of the present study was to evaluate the effects of supplementing the freezing medium with butylhydroxytoluene (BHT) and melatonin (MEL) in frozen-thawed pig spermatozoa. With this purpose, six ejaculates coming from six separate boars were cryopreserved in traditional freezing medium (i.e. lactose/egg-yolk/glycerol; Control) supplemented with 1.0 mM BHT (BHT-1), 2.0 mM BHT (BHT-2), 0.01 μM MEL (MEL-1) and 1.0 μM MEL (MEL-2). We evaluated sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidation of thiol groups, and levels of total reactive oxygen species (ROS), peroxynitrite and superoxide anion (·O 2 - ). We also analysed total (TM) and progressive sperm motilities (PM), and kinetic parameters at post-thaw (T0, T30 and T60). The BHT-2 and MEL-2 groups presented higher viability and acrosome integrity, and lower levels of peroxynitrite, ·O 2 - and lipid peroxidation than the control (P < 0.05), whereas MEL-2 diminished the levels of total ROS (P < 0.05). TM and PM were not affected by the treatment, while, LIN and STR shows differences between experimental groups. In conclusion, the addition of BHT and MEL to cryopreservation medium diminishes oxidative and nitrosative stress markers, which has repercussions for the integrity of plasma and acrosomal membranes of frozen-thawed spermatozoa., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this study, the morphology and ultrastructure of Genypterus blacodes spermatozoa were characterized through scanning and transmission electron microscopy. Findings revealed that the G. blacodes spermatozoa can be differentiated into three major parts: a spherical head without an acrosome (typical for externally fertilizing fish), a short mid-piece, and a long flagellum. The mean length of the spermatozoa was 57.6 ± 6.08 μm, with flagella accounting for 56.2 ± 7.2 μm. The head was 1.47 ± 0.2 μm long, and 0.89 ± 0.06 μm wide. The mid-piece had a total dimension of 0.72 ± 0.16 μm, and was 0.31 ± 0.02 μm in length and 0.6 ± 0.05 μm in width. It was located lateral to the nucleus and contained 4 or 5 spherical mitochondria. The mitochondria were separated from the axoneme by a cytoplasmic canal. The main piece of the flagellum had short irregular side-fins, and the axoneme was composed of the typical 9 + 2 microtubular doublet structure enclosed by a cell membrane. The present study reveals that G. blacodes sperm can be categorized as a primitive type. This study is the first to provide comprehensive details on the morphology and ultrastructure of spermatozoa in G. blacodes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We report the first case of a healthy baby born after intrauterine insemination with vitrified swim-up spermatozoa from an oligoasthenozoospermic patient. A 39-year-old patient was subjected to intrauterine insemination with spermatozoa from her 35-year-old husband, diagnosed with oligoasthenozoospermia. The swim-up spermatozoa from 2 ejaculates were suspended in a culture medium supplemented with 1% human serum albumin and 0.25 M sucrose. Three hermetically packaged 100-μL sperm portions (each containing 1.0 × 10(6) spermatozoa/mL) were vitrified by direct plunging into liquid nitrogen. The presence of leukocytes in the ejaculates was determined by indirect immunofluorescence assay. Production of reactive oxygen species was measured by chemiluminescent assay. Before intrauterine insemination, all portions were warmed in culture medium at 37°C, and the spermatozoa were concentrated by centrifugation; they were then resuspended in 500 μL of culture medium and inseminated. The cell suspension used for insemination displayed 60% progressive motility. Clinical pregnancy was confirmed at 7 weeks of gestation, and a healthy baby was born at term. Our results show that a successful intrauterine insemination can be achieved with aseptic vitrification of spermatozoa.

The aim of this study was to develop and to test the standardized aseptic technology of permeable cryoprotectant-free vitrification of human spermatozoa in capillaries (for intracytoplasmic sperm injection [ICSI] or in vitro fertilization [IVF]). To test the effect of vitrification on basic sperm parameters, each of 68 swim-up-prepared ejaculates from oligo-astheno-terato-zoospermic patients were aliquoted and distributed into 3 groups: 1) nontreated control, 2) 10 μL of spermatozoa cryopreserved by slow conventional freezing with glycerol-contented medium, and 3) 10 μL of spermatozoa vitrified in 50-μL plastic capillaries in culture medium with 0.25 M sucrose. Spermatozoa motility (1, 24, and 48 hours after warming), plasma membrane integrity, acrosomal integrity, and spontaneous capacitation-like changes were determined after warming. Aseptic cryoprotectant-free vitrification showed a significantly stronger cryoprotective effect compared with conventional freezing. One hour after warming, motility, plasma membrane integrity, and acrosomal integrity were significantly higher than is observed for conventionally frozen spermatozoa (28% vs 18%, 56% vs 22%, and 55% vs 21%, respectively; P < .05), although lower than in fresh spermatozoa (35%, 96%, and 84%, respectively; P < .05). Capacitation-like changes did not differ significantly between vitrified and conventionally frozen samples (8% vs 9%, respectively; P > .1) (2% in fresh spermatozoa). The newly developed technology of aseptic vitrification of human spermatozoa in capillaries can effectively preserve these cells from cryo-injures. Spermatozoa, vitrified by this technology, are free from seminal plasma owing to swim-up preceding vitrification and are free from permeable cryoprotectants. They are ready for further use immediately after warming without any additional treatment. Therefore, the reported technology has a great potential for use in ICSI/IVF.

Background: The aim of this study was to develop and to test the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume (for intrauterine insemination). Spermatozoa, vitrified by this technology, are free of permeant cryoprotectants and are ready for further use immediately after warming without any additional treatment (centrifugation or separation in the gradient for removal of cryoprotectant)., Methods: Each of 52 swim up-prepared ejaculates were divided into three aliquots and distributed into three treatment groups: Group 1: non-treated control; Group 2: spermatozoa cryopreserved by slow conventional freezing with glycerol-containing medium, and Group 3: spermatozoa vitrified in 0.5 mL insemination "French" straws in culture medium with 0.25 M sucrose. Sperm motility 1, 24 and 48 hours after warming, plasma membrane integrity and capacitation-like changes (spontaneous "cryo-capacitation" and acrosome reaction) were assessed after freezing-thawing., Results: In contrast to conventional freezing, spermatozoa vitrified with aseptic cryoprotectant-free technology displayed superior functional characteristics. The motility rate, integrity rates of cytoplasmic, and acrosomal membranes were significantly higher after vitrification than after conventional freezing (76% vs 52%, 54% vs 28% and 44% vs 30%, respectively) (p < 0.05). However, there were no differences between vitrification and conventional freezing in the presence of glycerol in terms of percentage of spermatozoa expressing CTC-capacitation pattern (11% vs 10%, respectively) (p > 0.1)., Conclusions: A basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.