In vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 °C–20 °C)

Accepted Manuscript In vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 to 20°C) Melina Andrea Formighieri Bertol, Romildo Romualdo Weiss, Luiz Ernandes Kozicki, Ana Claudia Machinski Rangel de Abreu, João Filipi Scheffer Pereira, Jonathan Jesus da Silva PII:

S0093-691X(16)30003-6

DOI:

10.1016/j.theriogenology.2016.03.030

Reference:

THE 13566

To appear in:

Theriogenology

Received Date: 22 October 2015 Revised Date:

25 February 2016

Accepted Date: 18 March 2016

Please cite this article as: Bertol MAF, Weiss RR, Kozicki LE, Abreu ACMRd, Pereira JFS, da Silva JJ, In vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 to 20°C), Theriogenology (2016), doi: 10.1016/ j.theriogenology.2016.03.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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In vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 to 20°C)

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Running title: FERTILIZATION OF CRYOPRESERVED EPIDIDYMAL BULL SPERM Melina Andrea Formighieri Bertol1,2, Romildo Romualdo Weiss2, Luiz Ernandes Kozicki3, Ana

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Claudia Machinski Rangel de Abreu2, João Filipi Scheffer Pereira3, Jonathan Jesus da Silva3 Department of Technology, Postgraduate studies in Bioprocess Engineering and Biotechnology,

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Human and Animal Health, Federal University of Paraná, Curitiba, Parana, Brazil. Department of Veterinary Medicine, Postgraduate studies in Animal Science, Pontifical

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Catholic University of Paraná, Curitiba, Paraná, Brazil.

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Correspondence: Melina Andrea Formighieri Bertol, Department of Veterinary Medicine, Federal University of Paraná, Funcionários Street, 1540, zip code 80035-050, Curitiba, Paraná, Brazil. E-mail: [email protected]

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ABSTRACT The aims of this study were to compare the viability and in vivo and in vitro fertilization potential post-thaw of sperm collected at different times post-orchiectomy from bull

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epididymides at 18 to 20°C, with those of semen collected by electroejaculation (EJ) from the same bulls. Semen samples (EJ) were collected from ten Zebu bulls and cryopreserved. A week later twenty epididymides from these bulls were obtained by orchiectomy and randomly divided

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into five groups (G) to be maintained at ambient temperature for 6, 12, 18, 24 and 30 h before sperm recovery by retrograde flow. The sperm were cryopreserved and after thawing parameters

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were determined by both computer assisted sperm analysis and morphological analysis. In vitro fertilization of oocytes was performed to assess the cleavage rate, blastocyst rate, total number of cells and hatching rate of embryos. The G30 sperm samples were also used for fixed time artificial insemination (FTAI) of Zebu heifers (n=10). The results of post-thaw sperm viability

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showed that total and progressive motility and plasma membrane integrity were lower in sperm in which cryopreservation was delayed for 30 h, showing a negative correlation of these parameters with delay before cryopreservation. In all groups it was possible to obtain viable

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embryos, and embryos from G6 samples had more cells than the other groups. The embryo production rate and hatching rate were significantly lower in G24 and G30 samples. For EJ many

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individual variations were observed in embryo production potential between bulls. G30 samples, with only 5.2% of post-thaw progressive motility, produced a pregnancy rate of 10% with FTAI. To the authors’ knowledge, this is the first time in vitro embryos up to eight days of development and a pregnancy after FTAI have been produced with sperm from bull epididymides that had been stored at 18 to 20°C for up to 30 hours. Keywords: Cryopreservation; epididymis; fertilization; embryo; sperm.

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1. Introduction The recovery, preservation and use of epididymal sperm are essential tools to preserve

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genetic stocks of valuable domestic or wild animals [1–3] under adverse conditions [4] and also

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as an alternative source of gametes in cases of human infertility [5,6]. Previous studies have

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already demonstrated the viability of bovine spermatozoa collected directly from the tail of

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epididymis [7,8], but in most cases the gametes were obtained immediately after slaughter or

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castration, or from epididymides that had been refrigerated at 5°C for long periods [9,10]. Few

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studies [11,12] have reproduced the real and more frequent situation, of the need for gamete

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utilization, i.e., accident, death or inability to obtain spermatozoa in the conventional way, when

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structures are exposed to ambient temperature before preservation.

Cryopreservation is the most effective method for long-term preservation of genetic

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material from valuable breeding individuals. The protocols and diluents used for

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cryopreservation of conventional bovine semen are well established, but when working with

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semen extracted directly from the epididymis many challenges remain. The spermatozoa

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retrieved from the tail of epididymis have special features, such as the absence of seminal plasma

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and large numbers of distal cytoplasmic droplets, which necessitate special handling, both for

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cryopreservation and in vitro fertilization [9,13]. Although it is a relatively new practice, good

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results have been achieved in cryopreservation of bovine epididymal spermatozoa using TRIS-

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based diluents containing egg yolk, glycerol and citric acid [1,8,10,14].

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After cryopreservation, the gametes can be used in biotechnologies such as artificial

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insemination and in vitro fertilization. The in vitro production of embryos is an indispensable

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biotechnology in mass propagation of genetic material since the number of embryos produced is

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far greater than those produced in vivo, and it allows genetic material from sub fertile females, of

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high genetic value, high livestock production, at different ages or reproductive status or even

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after death to be used [15]. Although production of in vitro embryos using cryopreserved

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spermatozoa obtained from bovine epididymides and stored at 5°C for long period of time [9]

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has previously been demonstrated, there are no reports of the fertilization potential of gametes

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retrieved from epididymides kept at average ambient field temperatures. In this context, the aims

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of this study were: to cryopreserve and assess the post-thaw viability of recovered sperm from

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the epididymides (EP) of zebu bulls, that had been kept at 18 to 20°C, and to assess potential for

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in vitro fertilization in vitro embryo production by evaluation of the cleavage rate, number of

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blastocysts, hatching rate and the number of embryo cells after fertilization with sperm from the

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epididymides (EP) and ejaculates (EJ) of the same bulls and also to determine the in vivo

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potential of fertilization in FTAI of heifers.

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2. Material and methods

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Animals in this study were used in accordance with all necessary recommendations and

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guidelines and approval of the Ethics Committee on Animal Use (CEUA-SCA/ UFPR, number

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017/2013) was obtained. All in vitro procedures were approved by the Ethics Committee on

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Animal Use (CEUA/PUCPR, number 894/2014).

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2.1 Animal Selection and ejaculate samples

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Pure Zebu bulls (Bos taurus indicus) of the Tabapuã breed (n=10) with an average age of

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63 months and weight 560 kg, from a beef cattle farm (25º37’0.4.4’’south, 52º48’58.9’’west, and

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at 505 m above sea level) were selected on the basis of a general clinical examination and a

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breeding soundness examination. The bulls were kept in an extensive grazing system, with grass

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(Cynodon spp) divided into paddocks and access to shelter, water and mineral salt ad libitum.

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Ejaculate samples (EJ) were collected by electroejaculation as this was the usual technique for

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semen collection. Semen samples were collected in the spring. The electroejaculation device (TK

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800) was introduced into the rectum and repeated electrical stimulation with direct current from

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zero to 780 mA for 3 s with three seconds intervals was applied until semen emission occurred.

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Semen was collected into a sterile graduated tube. Prioritization was given to minimizing

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discomfort. Two ejaculations were performed on each bull at three day intervals to remove

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sperm stored in the epididymis and a third ejaculate was collected a further three days later for

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inclusion in the study. The semen samples were evaluated for: subjective analysis of motility (0

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to 100%) – the average score during microscopic examination by two different evaluators, and

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sperm concentration, morphology and acrosomal defects. The sperm concentration was

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determined using a hemocytometer 1:100 dilution (semen:buffered saline-formalin solution) and

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morphology assessed on smears stained with Congo red.

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2.2 Obtaining testis and epididymis samples

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A week after obtaining the ejaculate, bilateral orchiectomy was performed under local

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anesthesia with 2% lidocaine without epinephrine. Skin and deeper tissue layers were incised and

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blood vessels and spermatic cord were clamped, the testes and epididymis were removed and

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immediately taken to the laboratory at the farm and randomly divided into five groups (n=4).

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Each group was maintained at 18 to 20°C for a variable period of time: 6, 12, 18, 24 and 30 h

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(G6, G12, G18, G24, G30). The temperature of 18 to 20°C was chosen as it is close to the

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average annual temperature in region where the study was conducted. Sperm was recovered from

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the epidymides by retrograde flow [16]. Each cauda epididymis was washed with 20 mL of a

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Botu-Turbo skimmed milk diluent (Botupharma) warmed to 37°C with its osmolarity specific for

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bovids (20 mL of distilled water to 100 mL of medium). After sperm harvest fresh samples were

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subjectively evaluated as described for ejaculate samples in section 2.1. The total motility ranged

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from 67.5% to 41.25% for G6 and G30 respectively, and the concentration of sperm per mL

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ranged from 426.8x106 (G12) to 101.8x106 (G30). The percentage of morphological defects and

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acrosome integrity were considered normal for epididymal sperm. A final volume of 20 mL

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(recovery medium and gametes) from each sample was centrifuged at 600 x g for 10 min to

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separate washing diluent and other contaminants such as blood and dirt.

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2.3 Cryopreservation

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After centrifugation the supernatant was discarded and the pellet resuspended with the

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extender Botu-bov (Botupharma) consisting of Tris-egg yolk, and 7% glycerol as cryoprotectant.

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The pre-freezing parameters of motility and the total cell number were assessed to verify the

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effects of centrifugation and diluent changes. Straws were filled with a concentration of 20

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million viable spermatozoa per 0.25 mL. The straws were sealed with polyvinyl alcohol and, for

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temperature stabilization, the doses were maintained for three hours in semen cooling container

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(5°C), and then placed horizontally on a 6 cm high support in an expanded polystyrene box

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containing liquid nitrogen for 20 min. Finally straws were immersed in liquid nitrogen at the

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storage temperature of -196°C. Forty doses from each bull of each group were cryopreserved.

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2.4 Sperm Evaluation

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Two straws from each bull, one EP and EJ, were thawed in a water bath at 40°C for 20 s

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and evaluated by computer analysis of semen (HTMA - Hamilton Thorn Motility Analyzer -

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IVOS 12.3). Three sample fields were chosen at random for assessment of: total motility (MT%);

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progressive motility (MP%); path velocity (VAP, um/s-1); straight-line velocity (VSL, um/s-1);

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curvilinear velocity (VCL, um/s-1); amplitude of lateral head displacement (ALH, mM); beat

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cross frequency (BCF Hz); straightness (STR%); linearity (LIN%); and rapid cells (RAP%). The

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percentage of morphologically deformed spermatozoa was determined by counting and

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classifying 200 cells using phase-contrast microscopy of wet mounts, and plasma membrane

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integrity was assessed using carboxyfluorescein diacetat (cFDA) and propidium iodide (PI)

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fluorescent dyes according to the methods previously described [17].

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2.5 In vivo fertilization

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The potential for in vivo fertilization of sperm from the epididymis was assessed using

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heifers at 36 ± 6 months of age, body condition score 3.5 from the same farm. Heifers were kept

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in pastures consisting mainly of Brachiaria sp., with mineral salt and water ad libitum. Ten

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Tabapuã heifers (Bos taurus indicus) were synchronized for fixed time artificial insemination

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(FTAI). Naturally cycling females were preferentially selected for the study. Each heifer was

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given 2 mg estradiol benzoate im and 1 g progesterone by vaginal device on D0, and on D8 150

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µg D-cloprostenol im, 300 IU eCG im, 1 mg estradiol cypionate im were given prior to removal

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of the intravaginal device . The FTAI was performed 50 h later. All hormones were obtained from

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Biogenesis Bagó, Argentina. Due to the small number of heifers available efforts were focused

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on estimating worst fertility. Sperm samples were selected from G30 (the group with the longest

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delay before harvest), with the worst post-thaw sperm parameters compared to the others.

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Pregnancy diagnosis was carried out 33 days after insemination by transrectal palpation and

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ultrasonography (Carewell CUS-3000V).

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2.6 In vitro production of embryos (IVPE)

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In this study, in vitro fertilization (IVF) techniques were used to verify the fertilizing

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capacity of epididymal bull sperm. Sperm sample from the epididymis (EP) of bulls in each

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group (6, 12, 18, 24 and 30h) were chosen by individual evaluation of bulls with the best post-

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thaw sperm parameters, in particular good progressive motility. The semen collected by

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electroejaculation (EJ) from the same bull was used as a control for all in vitro procedures.

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2.6.1 Collection and in vitro maturation of oocytes Cumulus-oocyte complexes were manually aspirated from medium antral follicles (3 to 8

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mm in diameter) on bovine ovaries obtained from a slaughterhouse within 5 hours of slaughter,

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using a hypodermic needle attached to a disposable 5 mL syringe (Becton Dickinson). After

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aspiration, oocytes and follicular fluid were transferred into a sterile petri dish to be selected,

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under a stereomicroscope. Selection was based on assessment of the characteristics of cumulus

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cells (absence, presence and number of layers) and the homogeneity of the ooplasm, according to

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the classification previously described [18]. Only high quality oocytes (category 1 and 2) were

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selected and transferred to a 35 mm diameter Petri dish where they were treated with 7 drops of

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80 uL IVM medium and placed under neutral mineral oil, in samples of 25 to 30 oocytes per

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drop. A total of 1,641 oocytes were used. In vitro maturation of oocytes was performed in TCM-

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199 medium (Sigma-Aldrich) containing 0.06849 mM/mL of glutamine, 10% fetal bovine

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serum-FBS (GIBCO), 22 mg/mL pyruvate, 0.5 µg/mL follicle stimulating hormone (FSH), 10

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IU/mL luteinizing hormone (LH), 1µg/mL estradiol and 0.1mg/mL amikacin sulfate for 24 h in a

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5% CO2 in air incubator at 38.7ºC. All culture media for the in vitro production of embryos were

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obtained from Sigma-Aldrich.

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2.6.2 Sperm Selection and in vitro fertilization

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To determine which sperm samples would be used for in vitro fertilization, samples were

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selected from the bulls in each group that showed the best sperm movement parameters,

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particularly progressive motility after thawing. The selected samples were thawed in a water bath

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at 35°C for 30 s. Then, due to the high percentage of medial and distal cytoplasmic droplets on

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sperm, the samples were maintained in a water bath (30°C) for 3 time periods (0, 5 and 20 min)

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to establish when spontaneous release of droplets occurred [19]. After centrifugation at 880 x g

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for 12 min, the pellet was resuspended in IVF. The medium and an spermatozoa aliquot was

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analyzed by optical microscopy at X 40 magnification for subjective assessment of the

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percentage of spermatozoa with progressive motility. Sample maintenance at 30°C for 20 min

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resulted in the highest percentage of spermatozoa with progressive motility, and fewer

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cytoplasmic droplets (and consequently more efficient separation of the pellet), and therefore this

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protocol was used in the final analysis. Samples from each group (G6, G12, G18, G24 and G30)

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from the epididymis (EP) and ejaculates (EJ) were thawed for 30 s at 35°C and maintained in a

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water bath at 30°C for 20 min. They were then transferred into polypropylene microtubes

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(Eppendorf) containing 400 µL of Bovipure (Nidacon International Laboratories AB) and

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Sperm-TALP in 1:1 ratio providing a gradient for sperm separation into two fractions (50 and

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100%). Centrifugation was performed at 880 x g for 12 min to separate the pellet containing

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spermatozoa with progressive motility, adapted from [20]. The pellet was aspirated using a

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micropipette and diluted in IVF medium containing Fert-TALP supplemented with 0.6 g/mL of

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fatty acid-free bovine serum albumin (FAF-BSA), 22 µg/mL pyruvate, 440 µg/mL PHE, 10

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IU/mL heparin and 1 mg/mL amikacin sulfate. After dilution, the spermatozoa were evaluated to

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determine the percentage of motile sperm (greater than 50% considered satisfactory). The

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solution containing IVF medium and a minimum concentration of one million spermatozoa per

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mL was transferred to each droplet containing 25 matured oocytes and incubated for 22 h under

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the same conditions as mentioned above at 2.6.1.

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2.6.3 In vitro Culture (IVC) and evaluation of embryos

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After fertilization, zygotes from each group were washed and transferred to the droplets

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containing CR2 medium supplemented with 0.5 g/L BSA-FAF, 5% BFS, 0.00034 mM/mL

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glutamine, 0.1 mM/mL of alanine and 0.1 mM/mL glycine and 0.1 mg/mL of amikacin sulfate

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and incubated in 5% CO2 in air at 38.7ºC for eight days. During cultivation, the embryos were

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evaluated on day three (D3) after fertilization to determine the cleavage rate, on day seven (D7)

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to count the number of blastocysts and on day eight (D8) to determine the rates of total

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blastocysts and the number of hatching embryos compared to the number of blastocysts observed

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on D7.

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On D8 of culture, the hatched embryos were separated for total cell count (CTC). They

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were transferred into solution containing 1 x TBS-Triton in 100 µL droplet for five min and then

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deposited into 60 µL droplets of DAPI (Sigma-Aldrich, USA) at a concentration of 1µL/mL for

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10 min. They were then placed between slides and coverslips containing 10 µL of glycerol

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(Sigma-Aldrich, USA). Fluorescence images were captured with a Leica DM4000 microscope

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and the images were processed and analyzed using LAS-AF software. Objectively, after

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evaluation of individual cell nuclei, the total number of cells from each embryo was computed.

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2.7 Statistical Analysis

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Statistical analysis of sperm viability was performed using the Shapiro-Wilk normality

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test. When data were not normally distributed, the Kruskal-Wallis test followed by Dunn's post-

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test was used. Analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison

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test was carried out to compare the data of sperm viability and in vitro production of embryos

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that were normally distributed. The data were transformed to r2 when there was no homogeneity

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of variance. A significance level of P<0.05 was assumed for all analyses. The JMP v.15 software

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(SAS Institute Inc., Cary, NC, USA) and Statgraphics Centurion XVI software were used to

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perform the statistical analysis.

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3. Results The post-thaw data of bovine epididymal spermatozoa at different time periods, shown in

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Table 1, indicate that total motility decreased as post-orchiectomy time increased, such that the

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total motile spermatozoa in G30 was lower than in G18 and G6 (P<0.05). Similarly the

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percentage of sperm with progressive motility was also influenced by the delay in preservation

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because the sperm that remained in the epididymal tail for 30 h showed significantly lower mean

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values of post-thaw progressive motility. The other parameters of sperm motility evaluated in

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CASA, such as VSL, ALH, BCF, STR e LIN did not differ between the groups (P>0.05). G30

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and EJ samples had lower percentages of rapid cells than G18 samples but were similar to the

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samples from the G6, G12 and G24.

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As can be seen in Table 1, the spermatozoa collected directly from the tail of epididymis

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had a higher percentage of altered morphology (P<0.05) compared to those collected by EJ.

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These abnormalities included the presence of a large number of distal and medial cytoplasmic

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droplets, which is considered a physiological change. Similar changes were seen in both fresh and

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pre-freeze sperm samples. Assessment of membrane integrity showed that epididymal sperm had

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a higher percentage of cells with intact membranes after thawing than EJ, except for G30

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samples, which were similar to EJ, with fewer spermatozoa with membrane-disruption.

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Data from the in vitro production of embryos with spermatozoa collected by

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electroejaculation (EJ) and directly from the epididymis tail (EP) at different post-orchiectomy

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periods (6, 12, 18, 24 and 30 h) from the same bulls (Table 2) are represented by the rates (%) of

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cleavage and blastocyst formation at D7 and D8. In all groups it was possible to produce viable

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embryos in vitro using both the semen collected by electroejaculation and the epididymal sperm

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from bulls up to 30 hours after orchiectomy. No statistical difference was observed when the

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results of cleavage between EJ and EP in different groups were compared, with the exception of

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G30, which showed higher cleavage rates (P<0.05) for EJ than EP samples. This group (G30)

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also showed higher blastocyst rates (P<0.05) in EJ on D7. The opposite was observed in G12,

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since the blastocyst rate on D7 and D8 was significantly greater when using spermatozoa that

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had remained for 12 hours in the epididymis tail. G24 showed low rates for all parameters

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evaluated with no differences (P>0.05) between EJ and EP.

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When embryo-production parameters were compared between epididymal (EP) groups, it

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was observed that G30 showed lower cleavage rates compared to G6 and G18. On D7 and D8,

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the blastocyst rate was lower (P<0.05) for G24 and G30 compared to the others. Comparing

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individuals (EJ), it can also be observed in Table 1 that the bull from G24 showed a significantly

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lower rate of cleavage and blastocyst on D7 than the bull from G30. The bull from G12 showed a

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lower production rate of blastocysts on D7 in comparison with G30. On D8, the blastocyst rate

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was significantly higher for the individuals from G30 and G6 compared to those from G12 and

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G24. In general the G12 and G24 bulls had the poorest performance in embryo production.

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Comparing the data in Table 3, which were obtained eight days after in vitro fertilization

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and the hatching of embryos produced post-fertilization with EJ and EP sperm, statistically

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significant differences were found only in G18, in which the embryo hatching rates were higher

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in EP than EJ. In the comparison between individuals, the individual bulls did not affect the

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hatch rate for EJ on D8. For EP, as well as other parameters assessed, the average hatch rate for

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G24 and G30 was significantly lower than for the other groups.

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It was only possible to compare the total cell counts of EJ and EP in G6 and G18. In G18

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no difference was observed in the mean number of cells per embryo (P>0.05) and in G6, higher

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mean numbers were seen in EP embryos than EJ's (P<0.05). When the groups were compared

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among themselves, there was no difference in the results of cell counts of embryos produced in

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EJ for any groups (P<0.05). Cell Count in EP was possible only in G6, G12 and G18, as there

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were insufficient number of embryos hatched in the other groups. The mean number of cells was

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greater in G6 than in G12 (P<0.05). The results of G18 were similar to those found in G6 and

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G12.

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Regarding in vivo testing of fertility potential after FTAI with post-thaw doses of G30,

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the sperm with 22.7% ± 12.1 of total motility and only 5.2% ± 3.3 of progressive motility were

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still capable of fertilization and a pregnancy was obtained (1/10).

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4. Discussion

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Although it is not possible to accurately control the variations in the ambient temperature

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to which bulls are exposed after sudden death, establishing a temperature range (18 to 20°C),

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close to the average annual temperature in most tropical regions, mimics real conditions. After

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successful cryopreservation of both epididymal sperm kept at 18 to 20°C before preservation and

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samples from ejaculation (EJ), assessment of sperm parameters post-thaw show that the time and

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temperature of storage had the biggest influence on results. The origin of the sperm was less

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important [21]. A previous study negatively correlated most sperm quality parameters with

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postmortem time when handling epididymal sperm of wild deer at 5ºC [22]. Changes in the

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tissue that occur after interruption of blood supply and autolytic processes affect the sperm,

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limiting its viability [23]. The longer the time spent in the epididymis at 18 to 20°C, the lower

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the total and progressive motility of sperm after cryopreservation, so G30 samples showed lower

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means for both parameters than the other groups. G6 and G18 samples showed significantly

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higher values than G30. Other authors have also observed that motility is the parameter most

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affected by post mortem or post orchiectomy time [22,12]. In addition to time of delay before

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processing, temperature and handling conditions also affect sperm viability [22,24]. At low temperatures (4-5°C), sperm tissue degradation is slower and sperm death

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delayed, so cryopreservation of sperm samples recovered from epididymides 72 h after

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orchiectomy in cattle [9] and up to 96 h in equine [25] are possible. These conditions do not

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reflect the reality in which an animal of high genetic value, or in danger of extinction, dies

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suddenly and is exposed to ambient temperature.

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Progressive motility of epididymal sperm was low in all groups, and this is partly due to

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the high number of distal cytoplasmic droplets present in the tail of the epididymal spermatozoa

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[19] which resulted in circular motion. The fresh, pre-freezing and thawed epididymal sperm of

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all groups had higher percentage of morphological alterations (P<0.05) than those obtained from

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the ejaculate. When sperm are obtained from the epididymis, the cytoplasmic droplets are

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considered to be physiological [26]. There was no difference in the percentage of cells with a

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damaged acrosome between the groups (P>0.05). The EJ and G30 groups had fewer spermatozoa

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with intact membranes after freezing compared to the other groups, showing higher cell fragility.

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The reduced fertility of frozen semen is largely attributed to the structure of the plasma

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membrane which has its function altered during freeze-thaw process [27]. Spermatozoa can

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maintain good progressive motility, and plasma membrane and acrosome integrity in the

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epididymis for up to three days if refrigerated (5°C) [9].

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In general, the percentage of plasma membrane integrity was lower in ejaculated sperm

273

(EJ) than in the epididymal samples (EP), with the exception of G30. The presence or absence of

274

seminal plasma affects the quality of post-thaw sperm. The total motility and integrity of the

275

acrosome and plasma membrane is better when the sperm is retrieved from the epididymis,

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without contact with seminal plasma, as compared to being harvested after ejaculation [28–30].

277

The seminal plasma can influence the fluidity and the lipid structure of the cell membrane,

278

making it more susceptible to damage during cryopreservation. Moreover there are variations in

279

the plasma composition between individuals that affects, to a greater or lesser degree, the

280

susceptibility to freezing [31].

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In this study, for the first time, in vitro embryos up to eight days of development were

282

produced after successful fertilization with cryopreserved sperm collected from epididymal

283

samples in all groups tested. This supports the future use of gametes even in non-optimal

284

temperature conditions. During in vitro production of embryos with epididymal sperm, the

285

blastocyst rate on D7 and D8 and the hatch rate on D8 were significantly lower in G24 and G30

286

samples compared to the others. This is not unexpected, since spermatozoa that remain in the

287

epididymis tail for longer before collection have their motility most affected, resulting in a lower

288

rate of sperm motility and therefore lower oocyte fertilization ability. The relationship between

289

good motility and fertilization capacity (and subsequently better embryo development) is well

290

known and was the sperm parameter that had the largest effect on success of in vitro embryo

291

production [32].

EP

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There was no ideal pellet formation for G24 and G30 samples. For these groups a low

293

percentage of spermatozoa showed progressive motility following centrifugation and therefore

294

small numbers were collected using a selective gradient. It was necessary to repeat

295

centrifugation, and even then the only small amounts of sperm exceeded the selection gradient.

296

The presence of cytoplasmic droplets and low progressive motility, due to post-orchiectomy time

297

and temperature were the factors that most hampered the isolation of epididymal sperm from

298

bulls for IVF. With sheep epididymides refrigerated within 24 h of slaughter more than 80% of

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spermatozoa with progressive motility could be recovered after selection with continuous

300

gradient Histoprep® and swim-up [33]. Another option for separation of epididymal sperm (as

301

demonstrated in the cat) is singe-layer centrifugation at 300 x g for 20 min with a colloidal

302

gradient and this seems to provide better results when compared to the swim-up method [2].

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The morphology of spermatozoa is also important for prediction of the potential for

304

fertilization, because most of the defects result in complete or partial exclusion during passage

305

through the female genital tract and prevent penetration of the zona pellucida [34]. The large

306

number of distal cytoplasmic droplets seems to have no influence on embryo production. The

307

protocol of thawing for 30 s and keeping the samples in a water bath for 20 min allowed the

308

spontaneous release of cytoplasmic droplets from the medial and distal region of the sperm tail.

309

Some authors report that proximal cytoplasmic droplets have a low impact on the efficiency of in

310

vitro embryo production, and that this is more affected by the interaction of morphological traits

311

and variations between breeding animals [13]. The results obtained among individuals,

312

demonstrate that there was a representative variation of the potential of in vitro fertilization of

313

each bull in EJ group, which may have influenced the results. Previous work also found

314

differences in in vitro production of embryos using semen from different bulls [13,35].

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Comparison of embryo production from EJ and EP for the same bulls showed that the

316

quality of semen and the percentage of sperm with progressive motility influenced the results of

317

the IVPE. In G30, the spermatozoa showed a significant drop in total motility, and there were

318

only 5.2% of cells with progressive motility in this group, impairing their fertilizing capacity,

319

resulting in lower cleavage rates on D3 and lower blastocyst rates on D7 compared to EJ from

320

the same animal. In G12, the blastocyst rate (D7 and D8) was greater for EP than EJ, and

321

epididymal sperm in this group showed better quality post-thaw. For European bull breeds

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(Holstein) no differences were found in IVPE of bovine blastocysts between the semen collected

323

by artificial vagina (ejaculate) and epididymal sperm of the same bulls when spermatozoa were

324

recovered immediately after orchiectomy [36]. The individual effect of the bull on embryo production was also observed for G24, where

326

poor results were seen in all parameters of IVPE with no difference between EP and EJ. It has

327

previously been reported that individual bulls influence the success of IVF, with great individual

328

variation in both post-thaw sperm parameters and embryo production among animals, and there

329

are bulls that are considered "bad freezers" and "bad producers of embryos" [13]. Our results

330

reinforce the importance of knowing the genetic background and the fertilization potential of

331

sperm donors in order to maximize success of IVF.

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Another factor that can influence the results of embryo production and fertilization is the

333

absence of seminal plasma. There are studies that relate the absence of seminal plasma, after

334

surgical removal of seminal vesicles in mice, with changes in motility and intrauterine transport

335

of sperm, reducing the oocyte fertilization rates [37]. Similarly, after removal of all male

336

accessory glands in rats there is a reduction in the cleavage rate of embryos and in vivo

337

implantation with significant embryonic loss [38]. In addition, indirect actions of seminal fluid

338

were identified in several factors that regulate embryonic development in the female

339

reproductive tract. The surgical excision of seminal vesicles in mice and consequently

340

ejaculation without seminal plasma can reduce fertility in females after mating. This occurs

341

because the number of embryos and the implantation rate is reduced, and the development of

342

viable blastocysts is slowed [39]. Despite the absence of seminal plasma in the EP samples, these

343

effects may have been reduced by immediate dilution of sperm from the epididymides with

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media containing substances that support sperm maintenance and survival during transit in the

345

female genital tract. Cell count is a noninvasive method for determining the quality of bovine embryos [40]

347

Proper assessment of blastocyst quality is essential in order to select the best embryos for

348

transfer. Hatched embryos in the epididymal group G6, with a higher average number of cell

349

nuclei, appear to be of highest quality (P<0.05). This group was superior even to its

350

corresponding group of EJ. Some groups, especially those with longer post-orchiectomy time

351

(G24 and G30, produced only a small number of hatched embryos on D8. Accurate evaluation of

352

blastocyst quality remains an important challenge for every embryologist when selecting the best

353

embryos for transfer. In emergency situations however all embryos should be used regardless of

354

the number and quality as they are the last gene pool of the donor.

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During ovarian harvest the presence of structures (medium follicles or corpus lutea) in

356

the cortical layer that indicated natural cycling were positively selected. Ovaries from

357

prepubescent females were avoided. After harvest oocytes were selected in the laboratory,

358

because the quality of oocytes also influences the results of in vitro fertilization oocytes without

359

or expanding cumulus cells were discarded. More embryos are produced from ovaries with more

360

cumulus cell layers and greater homogeneity of the oocyte cytoplasm [18].

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As there were a limited number of females for in vivo insemination, only G30 samples

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were used because this sperm group was exposed to greatest stress. Even with lower total and

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progressive motility, lower integrity of plasma membrane and increased delay in the epididymis

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before harvest, it was still possible to produce a pregnancy (1/10) after FTAI with G30 samples,

365

confirming its potential for in vivo fertilization. Although only 1 pregnancy occurred, this result

366

is very important since the production of a descendant of a high value animal after its death is a

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significant genetic gain and has high commercial value. The insemination after natural estrous

368

detection in cows with sperm that had been recovered from refrigerated epididymides (5°C)

369

resulting in two pregnancies [41]. This is the first report of insemination of estrus synchronized

370

heifers with sperm from bull epididymides kept at ambient temperature before sperm collection.

371

The ambient temperature of 18 to 20°C was chosen to mimic the annual average temperature in

372

the tropical region where the study was conducted. It would be interesting to repeat this research

373

in different weather conditions, testing higher and lower temperatures, reflecting the significant

374

variations in different regions.

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This study demonstrated that there is a window of opportunity in the field for recovery

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and utilization of genetic material from valuable domestic or wild animals after sudden death or

377

under other adverse conditions.

378

5. Conclusions

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In conclusion, after successful cryopreservation of epididymal sperm, the post-thaw

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parameters of total and progressive motility and plasma membrane integrity were most affected

381

the delay between orchiectomy time and preservation, being worse for G24 and G30 samples in

382

relation to samples from the other time periods. Following in vitro production of embryos, the

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total number of blastocysts at D7 and D8, and hatching rate at D8 were also lower in G24 and

384

G30 samples. The three factors that most influenced sperm viability and in vitro embryo

385

production were post-orchiectomy time, holding temperature of the epididymis, and individual

386

variations between bulls. After FTAI with G30 samples, one pregnancy occurred (1/10) although

387

the sperm had only 5.2% progressive motility. To the authors’ knowledge this is the first time

388

that embryos have been created in vitro and a pregnancy achieved using spermatozoa from

389

epididymides kept for up to 30 h at ambient temperature of 18 to 20°C.

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Acknowledgment

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The authors thank all the researchers and scientists from different institutions involved in this

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research, and the financial support granted by the National Counsel of Technological and

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Scientific Development (CnPQ).

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Chatdarong K, Thuwanut P, Morrell JM. Single-layer centrifugation through colloid selects improved quality of epididymal cat sperm. Theriogenology 2010;73:1284–92. doi:10.1016/j.theriogenology.2009.12.009.

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Fernández-Santos MR, Martínez-Pastor F, García-Macías V, Esteso MC, Soler AJ, de Paz P, et al. Extender osmolality and sugar supplementation exert a complex effect on the cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa. Theriogenology 2007;67:738–53. doi:10.1016/j.theriogenology.2006.10.005.

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Kaabi M, Paz P, Alvarez M, Anel E, Boixo JC, Rouissi H, et al. Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem. Theriogenology 2003:1249–59. doi:10.3923/ajava.2008.400.408.

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Drouineaud V, Sagot P, Faivre L, Michel F, Jimenez C. Birth after intracytoplasmic injection of epididymal sperm from a man with congenital bilateral absence of the vas deferens who had a robertsonian translocation. Fertil Steril 2003;79:1649–51. doi:10.1016/S0015-0282(03)00341-8.

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Martins CF, Rumpf R, Pereira DC, Dode MN. Cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production. Anim Reprod Sci 2007;101:326–31. doi:10.1016/j.anireprosci.2007.01.018.

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Martinez-Pastor F, Garcia-Macias V, Alvarez M, Chamorro C, Herraez P, De Paz P, et al. Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer. Theriogenology 2006;65:471–85. doi:10.1016/j.theriogenology.2005.05.045.

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Stojkovic M, Machado SA, Stojkovic P, Zakhartchenko V, Hutzler P, Gonçalves PB, et al. Mitochondrial distribution and adenosine triphosphate content of bovine oocytes before and after in vitro maturation: correlation with morphological criteria and developmental capacity after in vitro fertilization and culture. Biol Reprod 2001;64:904– 9. doi:10.1095/biolreprod64.3.904.

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Barth AD, Oko RJ. Abnormal Morphology of bovine spermatozoa. 1th ed. Iowa State: Iowa; 1989.

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Martínez AF, Martínez-Pastor F, Álvarez M, Fernández-Santos MR, Esteso MC, De Paz P, et al. Sperm parameters on Iberian red deer: Electroejaculation and post-mortem collection. Theriogenology 2008;70:216–26. doi:10.1016/j.theriogenology.2008.04.001.

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Martinez-Pastor F, Guerra C, Kaabi M, Diaz AR, Anel E, Herraez P, et al. Decay of sperm obtained from epididymes of wild ruminants depending on postmortem time. Theriogenology 2005;63:24–40. doi:10.1016/j.theriogenology.2004.03.003.

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Songsasen N, Tong J, Leibo SP. Birth of live mice derived by in vitro fertilization with spermatozoa retrieved up to twenty-four hours after death. J Exp Zool 1998;280:189–96.

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Tittarelli C, Savignone CA, Arnaudín E, Stornelli MC, Stornelli MA, de la Sota RL. Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides. Theriogenology 2006;66:1637–40. doi:10.1016/j.theriogenology.2006.01.021.

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Vieira LA, Gadea J, García-Vázquez FA, Avilés-López K, Matás C. Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fertilization capacity. Anim Reprod Sci 2013;136:280–8. doi:10.1016/j.anireprosci.2012.10.027.

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Guimarães T, Lopes G, Ferreira P, Leal I, Rocha A. Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells. Anim Reprod Sci 2012;136:85–9. doi:10.1016/j.anireprosci.2012.10.028.

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García-Álvarez O, Maroto-Morales A, Martínez-Pastor F, Garde JJ, Ramón M, Fernández-Santos MR, et al. Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection. Theriogenology 2009;72:160–8. doi:10.1016/j.theriogenology.2009.02.002.

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Stout MA, Saenz JR, Chenevert JF , Gentry GT, Bondioli KB, Godke RA. Criopreserved Ejaculated and epididymal sperm collected from the same holstein bulls used for in vitro fertilization. Reprod Fertil Dev 2012;25:261–261.

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Tables

MT (%) EJ

MP (%) ab

38.7 ± 24.8

RAP (%) a

12.8±11.1

19.2±15.8

EMD (%) a

a

26.1±7.6

56±9.4a

17.5±4.7ab

22.7±5.5ab

45±6b

G12

42.2±18.3ab

13±5.8ab

20.5±11.1ab

43±7.7b

G18

61±12.4a

23.7±9.3b

35.5±13.2b

42.2±2.2b

G24

51.2±13.3ab

15±2.5ab

24.2±7.3ab

44.5±1.9b

DA (%)

IMP (%)

a

12.8±4.5

27.2±10.5a

10.2±0.9ab

59.5±7b

9.5±1.2ab

63.7±9.7b

8±0.8b

56±11.2b

9.2±0.5ab

59.75±3.6b

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Table 1. Mean ± SD of Computer Assisted Sperm Analysis and morphological characteristics of post-thaw sperm collected by electroejaculation (EJ) and from epididymis (EP) of Tabapuã (Bos taurus indicus) bulls at different post-orchiectomy times (h) (G6, G12, G18, G24, G30).

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G30 22.7±12.1b 5.2±3.3a 9.25±7.1a 42±5.4b 10±0.8ab 32.2±4.9a Different letters in the same column indicate significant difference (P<0.05). MT = total motility; MP = progressive motility; RAP = rapid sperm cells; EMD = morphologically deformed sperm; DA = defects in acrosome membrane; IMP = plasma membrane integrity.

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Table 2. Total number of oocytes (n), mean ± SD of cleavage rate on day three, blastocyst on day seven (D7) accumulated blastocyst on day eight (D8), in vitro production of embryos using cryopreserved sperm collected by electroejaculation (EJ) and from epididymis (EP) of Tabapuã (Bos taurus indicus) bulls at different post-orchiectomy times (h) (G6, G12, G18, G24, G30). Blastocysts D8 (%) EJ EP aA 23.8±13.5 27.2±12.6aA 4.1±3.5bB 31.2±12.3aA 11.8±10.4abA 31.1±22.5aA 2.5±2.0bA 4.9±7.6bA 26.6±12.1aA 0.6±1.5bB line for the same

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Oocytes (n) Cleavage (%) Blastocysts D7 (%) EJ EP EJ EP EJ EP abA aA abA G6 159 180 67.5±9.9 69.1±12.6 15.0±8.5 20.9±14.6aA G12 170 159 57.5±18.7abA 64.6±11.9abA 2.4±3.1aB 24.1±7.2aA abA aA abA G18 156 157 55.2±18.3 70.8±14.0 10.5±10.7 21.3±15.0aA G24 159 133 47.1±7.4aA 51.6±20.1abA 1.9±2.1aA 3.2±4.4bA G30 186 182 74.5±13.2bA 40.9±15.8bB 19.7±7.0bA 0.0±0.0bB Different small letters in the column and different capital letters on the evaluation parameter indicate statistically significant differences (P<0.05).

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Groups

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Table 3. Embryonic hatch rate on day eight (D8) and total cell count (CTC) of the number of hatched embryos (n), produced in vitro using cryopreserved sperm collected by electroejaculation (EJ) and from epididymis (EP) of Tabapuã bulls (Bos taurus indicus) at different post-orchiectomy times (h) (G6, G12, G18, G24, G30). CTC (embryos number)

RI PT

Hatch D8 (%) Groups EP

EJ

G6

33.3±28.9aA

29.2±21.5aA

(13) 205.4aA

(15) 280.5aB

G12

11.2±19.5aA

39.6±26.3aA

(n/c)

(11) 191.7b

G18

10.2±19.7aB

43.9±20.7aA

G24

1.2±1.8aA

2.8±6.3bA

G30

38.5±41.6aA

EP

(8) 190.5aA

(15) 226.8abA

(n/c)

(n/c)

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EJ

(n/c)

4.3±5.6bA

(9) 225.4a

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Different small letters in the column and different capital letters on the line in the same parameter indicate statistically significant differences (P<0.05). In some samples the number of hatched embryos was insufficient, and was not counted (n/c).

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Highlights Alternative sources to obtain gametes are being studied for recovery genetic material.

•

It is important simulate more real conditions of handling gametes.

•

Sperm retrieval from epididymis 30 hours at environmental temperature from bulls.

•

Successful cryopreservation of sperm with good fertilization potential.

•

Never been described in scientific literature

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•