Papers by Heriberto Rodriguez-martinez
International Journal of Molecular Sciences, Apr 11, 2019
Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How th... more Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.
Cryobiology, Oct 1, 2014
Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for arti... more Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24 h at 15-17ºC and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24 h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24 h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
PubMed, 2001
The present study describes the use of a zona pellucida binding assay for the evaluation of canin... more The present study describes the use of a zona pellucida binding assay for the evaluation of canine spermatozoa. A zona pellucida binding assay is a sperm evaluation test that is practical to perform and provides potentially useful information on the damage caused to spermatozoa by new methods of sperm storage. The addition of the detergent Equex STM paste to the cryopreservation extender has a positive effect on the zona pellucida binding capacity of cryopreserved spermatozoa. A large number of sperm-oocyte complexes need to be evaluated because of the variability in sperm binding capacity among oocytes. However, this does not constitute a major problem as canine oocytes can be stored before use in a zona pellucida binding assay and sperm-oocyte complexes can be fixed and stored until evaluation.
Cryobiology, Feb 1, 2018
Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A ret... more Post-thaw boar sperm motility is affected by prolonged storage of sperm in liquid nitrogen. A retrospective study.
Animal Reproduction Science, Aug 1, 2018
This study aimed to elucidate the effect of SP from post-SRF on boar sperm freezability and, in a... more This study aimed to elucidate the effect of SP from post-SRF on boar sperm freezability and, in addition, to determine the relevance of sperm itself to sustain cryopreservation, regardless of the SP surrounding them. Twelve ejaculates from three boars were manually collected in fractions/portions, P1: the first 10 mL of the SRF, P2: the rest of the SRF and the post-SRF. Immediately, samples were centrifuged to separate spermatozoa from the surrounding SP. Spermatozoa from P1 and P2 were then incubated with its own SP or that from post-SRF, diluted in BTS (1:1, v/v) at 17 ºC overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P < 0.05) when P1-or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P < 0.05) regardless of the SP they were incubated in. Post-thaw sperm quality and functionality differed between P1 and P2, regardless of the SP used for overnight pre-freezing incubation. Post-thaw motility (P < 0.05) and viability (P < 0.01), as well as plasma membrane fluidity (P < 0.05) or lipid peroxidation values (P < 0.01) were best in P1 sperm compared to those of P2. The protein profile of sperm from P1 and P2, analyzed by 2D-PAGE, showed qualitative differences, which suggest that sperm rather than SP would explain differences in sperm freezability between ejaculate fractions/portions. Use of P1 fraction spermatozoa seems thus optimal for cryopreservation.
International Journal of Andrology, Dec 1, 2007
Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear wh... more Cryopreservation imposes dramatic changes in boar sperm survivability but it is as yet unclear which part of the process affects the spermatozoa the most. The present study monitored, along the entire process of cryopreservation, the stability (PMS) of the architecture of the lipid plasma membrane and its integrity (PMI), as well as the kinetics of the processed spermatozoa using two portions from the boar ejaculate (P1 ¼ the first 10 mL of the sperm-rich fraction, SRF; P2 ¼ the rest of the ejaculate), frozen in a recently developed package, the MiniFlatPack (MFPs, 0.5 • 10 9 sperm/dose). Evaluation was made at four specific stages, viz. S1 ¼ after collection (suspended in Beltsville thawing solution, BTS); S2 ¼ at 15°C (suspended in lactose-egg yolk, LEY); S3 ¼ at 5°C (suspended in LEY plus glycerol); and S4 ¼ post-thaw. Both sperm kinetics (using computer-assisted sperm analysis, CASA) and PMS [i.e. the degree of lipid disorder and of the exteriorization of phosphatidylserine (PS) in the plasma membrane, measured by flow cytometry using Merocyanine-540 (M-540), and Annexin-V (AV) respectively], as well as plasma membrane integrity [PMI, i.e. the degree of membrane damage, measured using Yo-Pro-1 or propidium iodide (PI)] were assessed after incubation in BTS at 38°C. Moreover, spermatozoa were challenged by incubation in modified Brackett-Oliphant medium (mBO+) with 37 mm of bicarbonate at 38°C for 30 min, and their PMS and PMI further explored. Total sperm motility was significantly higher in P1 than in P2 along the entire process (S1-S4; p < 0.01), decreasing significantly at S4 for both fractions (p < 0.0001). The proportion of spermatozoa showing linear motility (LinM) was similar between ejaculate portions (P1 and P2), with a significant increase post-thaw (S4; p < 0.0001). During cooling (S1-S3) but not post-thaw (S4), lateral head displacement (LHD) differed between portions and changed along the stages (p < 0.01). Sperm velocity differed between portions in S1 (p < 0.01), but remained similar, independently of the portion, thereafter (S2-S4). Both PMS and the total number of live spermatozoa remained similar between S1 and S3 while incubated in BTS for both ejaculate portions. Sperm mortality increased post-thaw (S4) in both portions but the degree of lipid disorder remained low in the live cells (1.28% for P1; 1.55% for P2). Exposure to mBO+, on the other hand, significantly increased membrane lipid disorder along cooling (S1-S3; p < 0.0001), increasing the percentages of dead spermatozoa, especially postthaw (around 70%, both portions). PS-exteriorization (AV) was not evident along the cryopreservation process in control (BTS) samples and exposure to
Animal Reproduction Science, Jun 1, 2016
Non-viable sperm ("dead sperm") are present in variable numbers in mammalian ejaculates and their... more Non-viable sperm ("dead sperm") are present in variable numbers in mammalian ejaculates and their number increase substantially when semen is stored, particularly cryopreserved. This review comparatively highlights, with experimental data in porcine, the role-played by non-viable sperm in the outcome of semen used in assisted reproductive technologies. As well, the review discusses our current understanding of their origin and the pathways involved when their large numbers negative influence the functional lifespan of contemporary viable sperm to eventually cause irreversible dysfunction that reduces their fertility potential and their ability to develop healthy embryos. Finally, it highlights procedures currently available to mitigate these harmful effects.
InTech eBooks, Mar 9, 2012
Reproduction in Domestic Animals, Oct 1, 2019
This work aimed to compare the ability of Sperm Chromatin Structure Assay (SCSA ®) and Sperm-Ovis... more This work aimed to compare the ability of Sperm Chromatin Structure Assay (SCSA ®) and Sperm-Ovis-Halomax ® to detect DNA fragmentation in frozen-thawed ram sperm incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with estrous sheep serum (SOF-ESS) at multiple time points (0-240 min). Incubation in SOF-ESS had no significant effects on SCSA ® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm-Ovis-Halomax ® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques suggesting that SCSA ® and Sperm-Ovis-Halomax ® may quantify different types of DNA damage in ovine spermatozoa under these experimental conditions.
Veterinary Medicine International, 2011
The present paper highlights aspects of the cryopreservation of boar semen, a species with partic... more The present paper highlights aspects of the cryopreservation of boar semen, a species with particular large, fractionated ejaculates, and a cumbersome cryotechnology that had prevented its commercial application. With the dramatic increase of use of liquid pig semen for artificial breeding over the past decade, developments on cryopreservation alongside the routine use of stud boar semen for AI had been promoted. Recent advances in our laboratory, accommodating the best use of portions of the sperm-rich fraction of the ejaculate for cryopreservation of the sperm-peak portion (P1) and parallel use of the rest of the collected ejaculated spermatozoa, appears as a suitable commercial alternative.
Animal reproduction, 2013
Modern livestock breeding is basically dependent on the proper use of semen for artificial insemi... more Modern livestock breeding is basically dependent on the proper use of semen for artificial insemination (AI) of females and of other reproductive biotechnologies such as the production of embryos in vitro for embryo transfer (IVP). Both these techniques have made possible not only the wide dissemination of genetic material onto breeding populations but also enhanced the selection of best sires, owing to the development of better diagnostic techniques for sperm function and of preservation of seminal material over time. Although use of liquid semen cooled to room temperature, to intermediate temperatures (+16-20°C) or chilled (+5°C) dominates in different species, cryopreservation is preferred in bovine AI and it is advancing in other species by the design of new containers, freezing methods and the use of better insemination strategies. Techniques to separate the aliquot of most robust spermatozoa from an ejaculate have shown a renascent particularly for sires with low sperm quality, and technological advances in separating spermatozoa for chromosomal sex make the technique suitable for commercial use, following application of novel findings in sperm and seminal plasma (SP) diagnostics and function. Alongside, knowledge of the epigenome and signalling capabilities of the semen (sperm and SP) calls for further studies regarding transgene production via ICSI for IVP or AI.
Theriogenology, 1998
Undiluted uterine fluid from 20 Warmblood/Standardbred mares (5 to 14 yr old) was recovered by ab... more Undiluted uterine fluid from 20 Warmblood/Standardbred mares (5 to 14 yr old) was recovered by absorption to an intrauterine tampon. The mares were considered gynecologically healthy based on a clinical examination including uterine swabs for cytology and bacteriology as well as endometrial biopsy examinations. The protein profiles (SDS-PAGE) and concentrations of total protein, albumin, and immunoglobulins (Ig) A and G in the uterine fluid were examined and compared with the same proteins in serum. Major peaks were identified on the obtained protein profiles, and there was a clear similarity between the serum profiles and uterine fluid profiles. Variability in protein concentrations among mares was considerably larger in uterine fluid than in serum. Concentrations of the various proteins in uterine fluid were 44 to 56% of those in serum, except for IgA, which had a similar concentration in both serum and uterine fluid. Concentration of the proteins corresponding to peak No. 3 (molecular weight 60 to 71 kDa) in uterine fluid was higher (P< 0.05) in younger mares than in older ones. Parity had no effect on the recorded protein concentrations. The present study of gynecologically healthy mares showed that there is a large individual variation in the protein composition of uterine fluid. The results suggest that age, but not parity, may affect this composition, and indicate further that there is considerable transudation to the uterine cavity.
Reproduction Fertility and Development, 2002
The present study was conducted to evaluate the effect of cumulus cells on the in vitro maturatio... more The present study was conducted to evaluate the effect of cumulus cells on the in vitro maturation (IVM) and glutathione (GSH) synthesis of porcine oocytes cultured in the presence or absence of cysteamine under different oxygen tensions, and on their subsequent male pronucleus formation after in vitro fertilization (IVF). Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 45 h in modified TCM-199 supplemented with or without 150 microM cysteamine under a humidified atmosphere of 5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2. When cultured in medium supplemented with cysteamine under 20% O2 tension, the rates of COC maturation to the metaphase II (MII) stage were significantly higher than those of DOs (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). Regardless of the addition of cysteamine and oxygen tension, the rates of male pronucleus formation in COCs after IVM and IVF were significantly higher than in DOs (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). The GSH content of oocytes was significantly increased by the addition of cysteamine to the maturation medium (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05), with significantly higher GSH content in COCs than in DOs (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). However, the GSH content of COCs and DOs was not significantly different when cultured in medium without cysteamine. These results indicate that cumulus cells play an important role in nuclear maturation to MII, GSH synthesis in porcine oocytes cultured in the presence of cysteamine, and subsequent male pronucleus formation after IVF.
Theriogenology, 2002
Twenty-®ve mature Brahman (Bos indicus) extensively reared breeding bulls were clinically examine... more Twenty-®ve mature Brahman (Bos indicus) extensively reared breeding bulls were clinically examined and electroejaculated at monthly intervals for 13 months to study if testicular consistency (TC), scrotal circumference (SC), sperm motility and morphology show seasonal variation under tropical conditions. Changes in SC were positively related to body condition (BC) (b 0:7 cm, P < 0:001) and age (P < 0:01). These changes were, however, not associated with deviations in TC, sperm motility or morphology (P > 0:05). Sperm motility was higher in samples collected during the breeding season than in samples collected at other times (62 versus 52%, LSM, P < 0:01). The frequency of bent tails with cytoplasmic droplet entrapped¯uctuated between monthly ejaculates, (LSM range 3±21%, P < 0:05). However, there was no relationship between these¯uctuations and environmental temperature, rainfall or changes in BC, TC or SC of the bulls (P > 0:05). Other sperm abnormalities did not change signi®cantly during the study period. The absence of a relationship between any of the climatic variables studied and SC, TC and sperm motility or morphology, respectively, indicates that temperature is not a main factor in¯uencing reproductive performance in Brahman bulls in the tropics. On the contrary, the changes found in BC followed by variations in SC suggest that nutrition may be a major factor affecting seasonal variations in male reproductive parameters, especially testicular size, in these sires.
Theriogenology, 2001
There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environme... more There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theta intema of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.
Journal of Veterinary Medicine Series A, 2007
The aim of this study was to characterize growth and sperm production parameters in Ogaden bucks ... more The aim of this study was to characterize growth and sperm production parameters in Ogaden bucks fed a basal diet of hay and supplemented with agro-industrial by-products and Khat leftovers in Ethiopia. Thirty-five bucks with a mean (±SD) initial live body weight (BW) of 15.5 ± 1.5 kg were randomly assigned to one of four dietary treatments for a period of 13 weeks. Treatments consisted of native hay fed ad libitum (control; C), native hay supplemented with a 1% of BW agroindustrial by-products (treatment 1; T1), native hay supplemented with Khat (Catha edulis) leftovers at a rate of 1% of BW (treatment 2; T2) and Khat leftovers fed ad libitum (treatment 3; T3). Bucks fed on T1-T3 had higher BW, body condition score, scrotal circumference (SC), testicular width and testicular length, compared to controls (P < 0.05). Also, bucks in T1-T3 had higher sperm progressive motility, sperm concentration per ml and total number of spermatozoa per ejaculate compared to controls (P < 0.05). Between treatments, bucks in T3 recorded the highest BW (17.2 ± 0.16) and testicular size (21.1 ± 0.17 cm). Both testicular and epididymal weight and dimensions were significantly affected (P < 0.05) by supplementation compared to controls. Testicular size was positively correlated to live BW (r ¼ 0.53, P < 0.001). SC was positively correlated with ejaculate volume (r ¼ 0.37, P < 0.001), sperm mass activity (r ¼ 0.65, P < 0.001) and individual sperm progressive motility (r ¼ 0.40; P < 0.001). Supplementation with Khat leftovers induced the highest improvement in live BW, testicular size, semen production and sperm motility in Ogaden bucks and can possibly be considered as a feed supplement to enhance goat production under smallholder livestock farming system in Ethiopia.
Journal of Veterinary Medicine Series A, 1996
SummaryTwenty peripubertal Swedish cross‐bred boars were used to study the testicular differentia... more SummaryTwenty peripubertal Swedish cross‐bred boars were used to study the testicular differentiation and the maturation of spermatozoa (epididymal) between 100 and 180 days of age. In all animals the sex cords were small and solid at 100 days of age. Spermatogenesis started at approximately 115 days of age and was completed, i.e. the cellular organization of the seminiferous tubules indicated a sexually mature testis with normal spermatogenesis, at 180 days of age. By 125 days of age only a few spermatozoa were found in the cauda epididymidis. Furthermore, a high frequency of sperm morphological alterations were seen. The sperm concentration in the cauda increased with increasing age, and simultaneously the frequency of abnormal spermatozoa decreased. However, not all boars had a ‘mature’ semen picture in the cauda epididymis at 180 days of age.
Reproduction in Domestic Animals, 1995
Reproduction in Domestic Animals, 1999
In order to study the T!cell response after the introduction of semen into the uterine cavity\ th... more In order to study the T!cell response after the introduction of semen into the uterine cavity\ the distribution of helper T cells "CD3¦# and cytotoxic:suppressor T cells "CD7¦# was exam! ined immunohistochemically in endometrial biopsy specimens[ Endometrial tissue samples were obtained from 08 gynae! cologically healthy mares during oestrus\ both before and 5 or 37 h after deposition of a single dose of neat stallion semen[ An increase "p 9[93# in the numbers of helper T cells "CD3¦# was observed at 5 h after insemination^thereafter the number of CD3¦ cells decreased to pre!insemination values by 37 h after insemination[ No signi_cant variations in numbers of CD7¦ cells were recorded either 5 or 37 h after insemination[ There seems to be an early "5 h#\ recruitment of helper T cells into the equine endometrium after semen deposition\ which might be related to the activation of the endometritis!like reac! tion seen as part of the equine uterine immune defence system during oestrus[ Inhalt Verbreitung von T!Zellen im Endometrium von Stuten 5 und 37 Stunden nach Besamung Um die Anwort von T!Zellen nach uteriner Besamung zu stu! dieren\ wurde die Verteilung von t!Helferzellen "CD3¦# sowie der zytotoxischen Subpressions!T!Zellen "CD7¦# immuno! histologisch in Uterusbiopsien untersucht[ Die endometrialen Gewebsproben wurden von 08 gyna Ãkologisch gesunden Stuten wa Ãhrend des O Ýstrus\ vor der Besamung sowie 5 bzw[ 37 Stunden danach gewonnen[ Fu à r die einmalige Besamung wurde unver! du à nntes Hengstsperma verwendet[ Ein zahlenma Ã)iger Anstieg "p 9\93# wurde bei den T!Helferzellen "CD3¦# 5 Stunden nach Besamung festgestellt[ Danach sank die Zahl der CD3¦ Zellen bis 37 Stunden nach Besamung auf Werte wie vor der Besamung ab[ Fu à r die Zahl der CD7¦ Zellen wurden keine zahlenma Ã)igen Vera Ãnderungen\ weder 5 noch 7 Stunden nach Besamung\ festgestellt[ Es scheint also eine fru à he "5 Stunden# Vermehrung von T!Helferzellen im Endometrium der Stute nach Besamung zu erfolgen[ Diese ko à nnte mit der Aktivierung einer Endometritis!a Ãhnlichen Reaktion als Teil des Immunab! wehrsystems wa Ãhrend des O Ýstrus bei der Stute in Zusammen! hang stehen[
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Papers by Heriberto Rodriguez-martinez