Dietary restriction in the form of fasting is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of long-term fasting and starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of fasting/starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected increased larval growth rate in F1 offspring from starved males and more malformed embryos at 24 hours post fertilisation in F2 offspring produced by F1 offspring from starved males. Comparing the transcriptomes of F1 embryos sired by starved and fed fathers revealed robust and reproducible increased expression of muscle composition genes but lower expression of lipid metabolism and lysosome genes in embryos from starved fathers. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal effects on extra-embryonic tissues which are loaded with maternal factors. We compared the embryo transcriptomes to published adult transcriptome datasets and found comparable repressive effects of starvation on metabolism-associated genes. These similarities suggest a physiologically relevant, directed, and potentially adaptive response transmitted by the father, independently from the offspring’s nutritional state, which was defined by the mother.

Animal model

We used wild-type Zebrafish Danio rerio from the AB strain obtained from ZIRC (Zebrafish International Resource Center, University of Oregon, Eugene, USA) and maintained at the SciLifeLab zebrafish platform at Uppsala University (http://www.scilifelab.se/facilities/zebrafish/) and the Controlled Ecology Facility (CEF) at the University of East Anglia (UEA, UK). The fish were kept in 3L tanks in a recirculating rack system (Aquatic Habitats (Uppsala) and Techniplast (UEA)) at 26.4 ± 1.4°C and a 12:12 diurnal light cycle.

Feeding regime

Fish were fed three times a day with a mixture of dry pellets and live artemia. For the experiments, 10-16 males were randomly split into 3L tanks assigned to the two experimental groups, referred to as Control and Starved. Each male was assigned an ID number and weighed and imaged for posterior identification through pigmentation and fin shape features. Males were maintained with wild-type companion females at a total fish density of 10 to 16 fish per 3l-tank for the duration of the experiment. For each experiment, we replicated the number of tanks ranging from a minimum of three up to seven tanks per treatment. The numbers vary for the different traits as not all fish always laid or produced gametes for IVF. Upon splitting into experimental groups, fish In the Starvation treatment were completely deprived of food while fish from the Control treatment were kept in the ad libitum feeding regime where they were fed three times a day under standard lab conditions. The starvation conditions were kept for 18 days. In zebrafish, a full cycle of spermatogenesis takes three weeks on average with the time between the last mitotic division and the sperm release into the spermatogenic tubule being six days (Leal et al., 2009). Here, the 18 days of starvation allowed exposure for one spermatogenic cycle.

In vitro fertilisation

On day 18, fish were put into breeding tanks and separated from wild-type females with dividers, allowing visual and olfactory contact. Breeding tanks were then covered with black cloths to avoid any light-induced oviposition the following morning (Westerfield, 2007).

Females and males were prepared for in vitro fertilisation and anaesthetised using 1.0–3.0 mg/l metomidate hydrochloride (AquacalmTM). Males were weighed to control for the effects of feeding regime and imaged to match their ID with the data collected on day 0 (Fed males n=49, Starved males n=49.). Males were then placed on a soft and wet sponge and squeezed gently in cranio-caudal direction to collect the ejaculate under a dissecting microscope (Nikon SMZ800). From each male, 0.2–0.8 μl of ejaculate was collected and transferred into a 0.2 ml Eppendorf tube containing 80 μl of Hank’s buffer (HBSS) and kept on ice for 5–10 min until IVF. Females were placed on 15 cm Petri dishes and gently stripped to obtain eggs. Clutches used for IVFs contained 20–300 high-quality eggs and they were used within one minute after stripping. To create the first generation (F1), we used a split clutch design to perform IVFs (Fig. 1A). Sperm samples were mixed gently, and each ejaculate and egg clutch were divided into two parts. In vitro fertilisation was performed simultaneously for both experimental groups. The sperm from each experimental male was used to fertilise split clutches from two different females when sperm availability allowed this second fertilisation. The sperm of males from seven independent tanks was used in seven rounds of IVF.

The reproductive success for each fish was assessed by analysing the number of fertilised eggs at 2 hpf(Fed fathers: 36.265 ± 24.870, n=3874; starved fathers: 41.592 ± 22.412, n=3947; data showed as mean ± SD), embryo survival at 24 hours postfertilization (hpf) (Fed males: 31.673 ± 23.112, n=1777; starved males: 35.3273 ± 21.697, n=2038; data showed as mean ± SD) and embryo defects at 2 (Embryos from fed males 6.020 ± 6.217; embryos from starved males 5.041 ± 4.247; data showed as mean ± SD) and 24 hpf (embryos from fed males 4.592 ± 5.697; embryos from starved males 6.265 ± 6.360; data showed as mean ± SD).

Eggs were considered unfertilised if there was no cell division within 2 hours after collection or were classified as dead if they were green or black in colour. Embryos were compared with diagrams describing the embryonic zebrafish development to study any abnormalities (Kimmel et al., 1995). Those embryos deviating from the diagrams were classified as abnormal. Embryos at 24 hpf were classified as dead if they had no yolk or showed abnormal black colour.

Natural spawning and reproductive fitness

The reproductive fitness of all experimental males (P) was studied by crossing them with non-experimental wild-type female fish from an independent AB population by natural spawning (Fed males n = 27, Starved males = 32.). One day before natural spawning, control and starved males were individually paired up with AB females in individual breeding tanks and were kept separated by a divisor, allowing for visual and olfactory contact. The next morning, the divisor was removed to let each pair spawn. Breeding tanks were checked every half hour and eggs were collected and transferred into Petri dishes containing a E3 and 0.1% methylene blue solution to avoid fungal growth. Plates were kept in an incubator set at 28°C.

Reproductive success for each experimental male in P was assessed by analysing the number of fertilised eggs (Fed males: 112 ± 53.778, n=2784. Starved males: 88.688 ± 42.815, n=1608; data showed as mean ± SD). The data was collected from 3 independent rounds of breeding with males from four separate tanks per treatment.

A standardised number of F1 offspring sired upon IVF with Fed or Starved were reared into adulthood for assessment of their reproductive fitness males (13.957 ± 8.544, from 23 Fed or Starved fathers). F1 larvae were kept in groups of 50 and, at the age of two months, fish were re-distributed into 3l tanks in mixed sex groups of 14-16 fish per tank (survival at 2 months in fish from fed fathers 10.21739 ± 8.179556, survival in fish from starved fathers 10.56522 ± 7.703754). F1 males and females were crossed by natural spawning with wildtype females or males, respectively, following the procedure described above.

Similarly to the P males, the reproductive success of F1 males was assessed analysing fertilised eggs (Fertilised eggs of females from fed fathers: 109 ± 59.556, n=944; fertilised eggs of females from starved fathers: 94.905 ± 64.098, n=2327; eggs fertilised by males from fed fathers: 114.167 ± 88.205, n=1398; eggs fertilised by males from starved fathers: 174.667 ± 103.645, n=535; showed as mean ± SD), embryo defects at 2 hpf (64-cells) (Number of abnormal eggs from offspring of fed fathers: 2.1 ± 2.732, n=2342. Number of abnormal eggs from offspring of starved fathers: 1.522 ± 2.538, n=2862; showed as mean ± SD), embryo survival at 24 hpf (live embryos from offspring of fed fathers: 71.75 ± 64.142, n=1435; live embryos from offspring of starved fathers: 80.292 ± 55.637, n=1927; showed as mean ± SD), and embryo defects at 24 hpf (abnormal embryos from offspring of fed fathers: 0.3 ± 0.571; abnormal embryos from offspring of starved fathers: 10.0 ± 29.329; showed as mean ± SD).

Embryo/larvae phenotype

Upon confirmation of fertilisation (2 hpf), we placed individual fertilised eggs onto 12-well plates for monitoring of embryos phenotypes which included:

Hatching rate every two hours from 48 hpf to 58 hpf (Healthy embryos from fed fathers at 48 hpf: 1.25 ± 2.81, embryos from starved fathers at 48 hpf: 1.643 ± 2.792, Embryos from fed fathers at 50 hpf: 6.357 ± 6.993, embryos from starved fathers at 50 hpf: 5.214 ± 5.62, Embryos from fed fathers at 52 hpf: 11.929 ± 8.366, embryos from starved fathers at 52 hpf: 11.214 ± 7.089, Embryos from fed fathers at 54 hpf: 16.179 ± 8.777, embryos from starved fathers at 54 hpf: 14.964 ± 7.1, Embryos from fed fathers at 56 hpf: 18.107 ± 8.456, embryos from starved fathers at 56 hpf: 17.25 ± 6.995, Embryos from fed fathers at 58 hpf: 22.261 ± 5.948, embryos from starved fathers at 58 hpf: 21.652174 ± 4.386; showed as mean ± SD). Hatching rate was defined as the number of larvae that hatched at each time point analysed out of the total number of live embryos.

Yolk utilisation at 24 hpf and 5 dpf (yolk diameter in embryos from fed fathers at 24 hpf: 0.6387 ± 0.0277 mm, yolk diameter in embryos from starved fathers at 24 hpf: 0.639 ± 0.026 mm, yolk area in embryos from fed fathers at 5 dpf: 0.00476 ± 0.00168 cm2, yolk area in embryos from starved fathers at 5 dpf: 0.0044 ± 0.00162 cm2, yolk length in embryos from fed fathers at 5 dpf: 3.825 ± 0.121 mm, yolk length in embryos from starved fathers at 5 dpf: 3.802 ± 0.139 mm)

Growth rate between 5 and 8 dpf (length of larvae from fed fathers at 5 dpf 3.819 ± 0.120 mm, length of larvae from starved fathers at 5 dpf 3.808 ± 0.139 mm, length of larvae from fed fathers at 8 dpf 4.277 ± 0.203 mm, length of larvae from starved fathers at 8 dpf 4.326 ± 0.174 mm).

Embryos from fed fathers included in the hatching rate analysis n=610, embryos from starved fathers n=611. For the yolk utilisation and the growth rate analyses, a picture of each embryo was taken under a dissection microscope at 24 hpf, 5 dpf and 8 dpf. The measurements were taken for yolk diameter at 24 hpf (Embryos from fed fathers n= 252, embryos from starved fathers n=258), larval length (Larvae from fed fathers n= 175, larvae from starved fathers n= 180), yolk length and lipid droplet at 5dpf (Larvae from fed fathers n= 70 , larvae from starved fathers n= 80) and larval length at 8dpf (Larvae from fed fathers n= 287, larvae from starved fathers n= 278).

Computer Assisted Sperm Analysis (CASA)

To assess sperm motility, we used Computer-Assisted Sperm Analysis (CASA; ISAS; Proiser, R+D, S.L.). We placed 2 µl onto a mixing plate which was activated with 3 µl of tank water at 28°C on a Cytonix 4 Chamber slide (MicroTool B4 Slide, 20 µm depth). A recording taken every ten seconds starting 10 seconds post activation until 60 seconds post activation. We recorded sperm movement using a brightfield microscope (UOP UB203i trinocular microscope; Proiser) at 100× magnification and a black and white video camera (782M monochrome CCD progressive camera; Proiser). The recordings were analysed using ISAS v1 software (Proiser) with the following settings: frame rate: 50 frames/s; frames used: 50; particle area: 5–50 μm2; threshold measurements for VCL: slow, 10–45 μm/s; medium, 45–100 μm/s; rapid, >100 μm/s (Alavioon et al., 2017).

Statistical analysis

All analyses were conducted using the statistical software R v. 4.1.2. Linear models (LM) were fitted using the lm function, linear mixed-effects models were applied using lmer function and Generalised Linear Mixed-Effects models (GLM) were applied using the glmer function from lme4 package on phenotypic data following a binomial response (e.g. embryo survival and fertilisation success). We included traits of interest as response variables, and treatment, time and sex where applicable as fixed effects with tank ID, Block and IVF round as random factor. We excluded random factors that explained no variation to avoid over-parametrisation of the models.

Data from: Paternal starvation affects metabolic gene expression during zebrafish offspring development and life-long fitness

Data files

Abstract

Description of the data and file structure

CSV File Name: F1_survival2months_sexratio.txt

CSV File Name: F1_yolkdiam_1dpf.txt

CSV File Name: F1fertsuccandSexDiff.txt

CSV File Name: Fert_Succ_naturalspawning.txt

CSV File Name: MaleWeight.txt

CSV File Name: F1_growth_data.txt

CSV File Name: Weight-ejac.quality_IVF.txt

CSV File Name: F1_hatching.txt

CSV File Name: F1_IVF_fertsucc.txt

CSV File Name: F1_lipid.txt

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